Random peptides that bind to gastro-intestinal tract (GIT) transport receptors and related methods

ABSTRACT

This invention relates to proteins (e.g., peptides) that are capable of facilitating transport of an active agent through a human or animal gastrointestinal tissue, and derivatives (e.g., fragments) and analogs thereof, and nucleotide sequences coding for said proteins and derivatives. The proteins of the invention have use in facilitating transport of active agents from the lumenal side of the GIT into the systemic blood system, and/or in targeting active agents to the GIT. Thus, for example, by binding (covalently or noncovalently) a protein of the invention to an orally administered drug, the drug can be targeted to specific receptor sites or transport pathways which are known to operate in the human gastrointestinal tract, thus facilitating its absorption into the systemic system.

This application claims priority to U.S. provisional application Ser.No. 60/046,595 filed May 15, 1997, which is incorporated by referenceherein in its entirety.

1. INTRODUCTION

The present invention relates generally to random peptides capable ofspecific binding to gastrointestinal tract (GIT) transport receptors. Inparticular, this invention relates to peptide sequences and motifs, aswell as derivatives thereof, which enhance drug delivery and transportthrough tissue, such as epithelial cells lining the lumenal side of thegastro-intestinal tract (GIT). Production of peptides, derivatives andantibodies is also provided. The invention further relates topharmaceutical compositions, formulations and related methods.

2. BACKGROUND OF THE INVENTION

2.1. Peptide Libraries

There have been two different approaches to the construction of randompeptide libraries. According to one approach, peptides have beenchemically synthesized in vitro in several formats. Examples ofchemically synthesized libraries can be found in Fodor, S., et al.,1991, Science 251: 767–773; Houghten, R., et al., 1991, Nature 354:84–86; and Lam, K., et al., 1991, Nature 354: 82–84.

A second approach to the construction of random peptide libraries hasbeen to use the M13 phage, and, in particular, protein pIII of M13. Theviral capsid protein of M13, protein III (pIII), is responsible forinfection of bacteria. Several investigators have determined frommutational analysis that the 406 amino acid long pIII capsid protein hastwo domains. The C-terminus anchors the protein to the viral coat, whileportions of the N-terminus of pIII are essential for interaction withthe E. coli pillin protein (Crissman, J. W. and Smith, G. P., 1984,Virology 132: 445–455). Although the N-terminus of the pIII protein hasshown to be necessary for viral infection, the extreme N-terminus of themature protein does tolerate alterations. In 1985, George Smithpublished experiments reporting the use of the pIII protein ofbacteriophage M13 as an experimental system for expressing aheterologous protein on the viral coat surface (Smith, G. P., 1985,Science 228: 1315–1317). It was later recognized, independently by twogroups, that the M13 phage pIII gene display system could be a usefulone for mapping antibody epitopes (De la Cruz, V., et al., 1988, J.Biol. Chem. 263: 4318–4322; Parmley, S. F. and Smith, G. P., 1988, Gene73: 305–318).

Parmley, S. F. and Smith, G. P., 1989, Adv. Exp. Med. Biol. 251: 215–218suggested that short, synthetic DNA segments cloned into the pIII genemight represent a library of epitopes. These authors reasoned that sincelinear epitopes were often ⁻6 amino acids in length, it should bepossible to use a random recombinant DNA library to express all possiblehexapeptides to isolate epitopes that bind to antibodies. Scott, J. K.and Smith, G. P., 1990, Science 249: 386–390 describe construction andexpression of an “epitope library” of hexapeptides on the surface ofM13. Cwirla, S. E., et al., 1990, Proc. Natl. Acad. Sci. USA 87:6378–6382 also described a somewhat similar library of hexapeptidesexpressed as gene pIII fusions of M13 fd phage. PCT Application WO91/19818 published Dec. 26, 1991 by Dower and Cwirla describes a similarlibrary of pentameric to octameric random amino acid sequences. Devlinet al., 1990, Science, 249: 404–406, describes a peptide library ofabout 15 residues generated using an (NNS) coding scheme foroligonucleotide synthesis in which S is G or C. Christian and colleagueshave described a phage display library, expressing decapeptides(Christian, R. B., et al., 1992, J. Mol. Biol. 227: 711–718).

Other investigators have used other viral capsid proteins for expressionof non-viral DNA on the surface of phage particles. For example, themajor capsid protein pVIII was so used by Cesareni, G., 1992, FEBS Lett.307: 66–70. Other bacteriophage than M13 have been used to constructpeptide libraries. Four and six amino acid sequences corresponding todifferent segments of the Plasmodium falciparum major surface antigenhave been cloned and expressed in the filamentous bacteriophage fd(Greenwood, J., et al., 1991, J. Mol. Biol. 220: 821–827).

Kay et al., 1993, Gene 128: 59–65 (Kay) discloses a method ofconstructing peptide libraries that encode peptides of totally randomsequence that are longer than those of any prior conventional libraries.The libraries disclosed in Kay encode totally synthetic random peptidesof greater than about 20 amino acids in length. Such libraries can beadvantageously screened to identify peptides, polypeptides and/or otherproteins having binding specificity for a variety of ligands. (See alsoU.S. Pat. No. 5,498,538 dated Mar. 12, 1996; and PCT Publication No. WO94/18318 dated Aug. 18, 1994.)

A comprehensive review of various types of peptide libraries can befound in Gallop et al., 1994, J. Med. Chem. 37:1233–1251.

Screening of peptide libraries has often been done 25 using an antibodyas ligand (Parmley and Smith, 1989, Adv. Exp. Med. Biol. 251:215–218;Scott and Smith, 1990, Science 249:386–390). In many cases, the aim ofthe screening is to identify peptides from the library that mimic theepitopes to which the antibodies are directed. Thus, given an availableantibody, peptide libraries are excellent sources for identifyingepitopes or epitope-like molecules of that antibody (Yayon et al., 1993,Proc. Natl. Acad. Sci. USA 90:10643–10647).

McCafferty et al., 1990, Nature 348:552–554 used PCR to amplifyimmunoglobulin variable (V) region genes and cloned those genes intophage expression vectors. The authors suggested that phage libraries ofV, diversity (D), and joining (J) regions could be screened withantigen. The phage that bound to antigen could then be mutated in theantigen-binding loops of the antibody genes and rescreened. The processcould be repeated several times, ultimately giving rise to phage whichbind the antigen strongly.

Marks et al., 1991, J. Mol. Biol. 222:581–597 also used PCR to amplifyimmunoglobulin variable (V) region genes and cloned those genes intophage expression vectors.

Kang et al., 1991, Proc. Natl. Acad. Sci. USA 88:4363–4366 created aphagemid vector that could be used to express the V and constant (C)regions of the heavy and light chains of an antibody specific for anantigen. The heavy and light chain V-C regions were engineered tocombine in the periplasm to produce an antibody-like molecule with afunctional antigen binding site. Infection of cells harboring thisphagemid with helper phage resulted in the incorporation of theantibody-like molecule on the surface of phage that carried the phagemidDNA. This allowed for identification and enrichment of these phage byscreening with the antigen. It was suggested that the enriched phagecould be subject to mutation and further rounds of screening, leading tothe isolation of antibody-like molecules that were capable of evenstronger binding to the antigen.

Hoogenboom et al., 1991, Nucleic Acids Res. 19:4133–4137 suggested thatnaive antibody genes might be cloned into phage display libraries. Thiswould be followed by random mutation of the cloned antibody genes togenerate high affinity variants.

Bass et al., 1990, Proteins: Struct. Func. Genet. 8:309–314 fused humangrowth hormone (hGH) to the carboxy terminus of the gene III protein ofphage fd. This fusion protein was built into a phagemid vector. Whencells carrying the phagemid were infected with a helper phage, about 10%of the phage particles produced displayed the fusion protein on theirsurfaces. These phage particles were enriched by screening with hGHreceptor-coated beads. It was suggested that this system could be usedto develop mutants of hGH with altered receptor binding characteristics.

Lowman et al., 1991, Biochemistry 30:10832–10838 used an improvedversion of the system of Bass et al. described above to select formutant hGH proteins with exceptionally high affinity for the hGHreceptor. The authors randomly mutagenized the hGH-pIII fusion proteinsat sites near the vicinity of 12 amino acids of hGH that had previouslybeen identified as being important in receptor binding.

Balass et al., 1993, Proc. Natl. Acad. Sci. USA 90:10638–10642 used aphage display library to isolate linear peptides that mimicked aconformationally dependent epitope of the nicotinic acetylcholinereceptor. This was done by screening the library with a monoclonalantibody specific for the conformationally dependent epitope. Themonoclonal antibody used was thought to be specific to the acetylcholinereceptor's binding site for its natural ligand, acetylcholine.

2.2. Drug Delivery Systems

The common routes of therapeutic drug administration are oral ingestionor parenteral (intravenous, subcutaneous and intramuscular) routes ofadministration. Intravenous drug administration suffers from numerouslimitations, including (i) the risk of adverse effects resulting fromrapid accumulation of high concentrations of drug, (ii) repeatedinjections which can cause patient discomfort; and (iii) the risk ofinfection at the site of repeated injections. Subcutaneous injection isnot generally suitable for delivering large volumes or for irritatingsubstances. Whereas oral administration is generally more convenient, itis limited where the therapeutic agent is not efficiently absorbed bythe gastrointestinal tract. To date, the development of oralformulations for the effective delivery of peptides, proteins andmacromolecules has been an elusive target. Poor membrane permeability,enzymatic instability, large molecular size, and hydrophilic propertiesare four factors that have remained major hurdles for peptide andprotein formulations (reviewed by Fix, J. A., 1996, J. Pharmac. Sci.85:1282–1285). In order to develop an efficacious oral formulation, thepeptide must be protected from the enzymatic environment of thegastrointestinal tract (GIT), presented to the absorptive epithelialbarrier in a sufficient concentration to effect transcellular flux (Fix,J. A., 1996, J. Pharmac. Sci. 85:1282–1285), and if possible “smuggled”across the epithelial barrier in an apical to basolateral direction.

Site specific drug delivery or drug targeting can be achieved atdifferent levels, including (i) primary targeting to a specific organ,(ii) secondary targeting to a specific cell type within that organ and(iii) tertiary targeting where the drug is delivered to specificintracellular structures (e.g., the nucleus for genes) (reviewed inDavis and Jllum, 1994, In: Targeting of Drugs 4, (Eds), Gregoriadis,McCormack and Poste, 183–194). At present there is a considerable amountof ongoing research work in the Drug Delivery Systems (DDS) area, andmuch of it addresses (i) targeting delivery and (ii) the development ofnon-invasive ways of getting macromolecules, peptides, proteins,products of the biotechnology industry, etc. into the body (Evers, P.,1995, Developments in Drug Delivery: Technology and Markets, FinancialTimes Management Report). It is generally accepted that targeted drugdelivery is crucial to the improved treatment of certain diseases,especially cancer, and not surprisingly many of the approaches totargeted drug delivery are focused in the cancer area. Many anticancerdrugs are toxic to the body as well as to malignant cells. If a drug, ora delivery system, can be modified so that it “homes in” on the tumor,then by maximizing the drug concentration at the disease site, theanti-cancer effect can be exploited to the full, while toxicity isgreatly reduced. Tumors contain antigens which provoke the body torespond by producing antibodies designed to attach to the antigens anddestroy them. Monoclonal antibodies are being used as both deliveryvehicles targeted to tumor cells (reviewed by Pietersz, G. A., 1990,Bioconjugate Chem. 1:89–95) and as imaging agents to carry molecules ofdrug or imaging agent to the tumor surface.

2.3. Transport Pathways

The epithelial cells lining the lumenal side of the GIT are a majorbarrier to drug delivery following oral administration. However, thereare four recognized transport pathways which can be exploited tofacilitate drug delivery and transport: the transcellular, paracellular,carrier-mediated, and transcytotic pathways. The ability of aconventional drug, peptide, protein, macromolecule or nano- ormicroparticulate system to “interact” with one of these transportpathways may result in increased delivery of that drug or particle fromthe GIT to the underlying circulation.

In the case of the receptor-mediated, carrier-mediated or transcytotictransport pathways, some of the uptake signals have been identified.These signals include, inter alia, folic acid, which interacts with thefolate receptor, and cobalamin, which interacts with Intrinsic Factor.In addition, leucine- and tyrosine-based peptide sorting motifs orinternalization sequences exist, such as YSKV, FPHL, YRGV, YQTI, TEQF,TEVM, TSAF, and YTRF (SEQ ID NOS:203, 204, 205, 206, 207, 208, 209, and210, respectively), which facilitate uptake or targeting of proteinsusing specific membrane receptors or binding sites to identify peptidesthat bind specifically to the receptor or binding site.

Non-receptor based assays to discover particular ligands have also beenused. For instance, a strategy for identifying peptides that altercellular function by scanning whole cells with phage display librariesis disclosed in Fong et al., Drug Development Research 33:64–70 (1994).However, because whole cells, rather than intact tissue or polarizedcell cultures, are used for screening phage display libraries, thisprocedure does not provide information regarding sequences whose primaryfunction includes affecting transport across polarized cell layers.

Additionally, Stevenson et al., Pharmaceutical Res. 12(9), S94 (1995)discloses the use of Caco-2 monolayers to screen a synthetic tripeptidecombinatorial library for information relating to the permeability ofdi- and tri-peptides.

A method of identifying a peptide which permits or facilitates thetransport of an active agent through human or animal tissues has beendeveloped (see U.S. patent application Ser. No. 08/746,411 filed Nov. 8,1996, which is incorporated by reference herein in its entirety). Phagefrom a random phage library is plated onto or brought into contact witha first side, preferably the apical side, of a tissue sample, either invitro, in vivo or in situ, or polarized tissue cell culture. The phagewhich is transported to a second side of the tissue opposite the firstside, preferably the basolateral side, is harvested to selecttransported phages. The transported phages are amplified in a host andthis cycle is repeated (using the transported phage from the most recentcycle) to obtain a selected phage library containing phage which can betransported from the first side to the second side.

Discussion or citation of a reference hereinabove shall not be construedas meaning that such reference is prior art to the present invention.

3. SUMMARY OF THE INVENTION

The present invention relates generally to random peptides and peptidemotifs capable of specific binding to GIT transport receptors. Suchproteins can be identified using any random peptide library, e.g., achemically synthesized peptide library or a biologically expressedpeptide library. If a biological peptide expression library is used, thenucleic acid which encodes the peptide which binds to the ligand ofchoice can be recovered, and then sequenced to determine its nucleotidesequence and hence deduce the amino acid sequence that mediates binding.Alternatively, the amino acid sequence of an appropriate binding domaincan be determined by direct determination of the amino acid sequence ofa peptide selected from a peptide library containing chemicallysynthesized peptides. In a less preferred aspect, direct amino acidsequencing of a binding peptide selected from a biological peptideexpression library can also be performed.

In particular, this invention relates to proteins (e.g., peptides) thatare capable of facilitating transport of an active agent through a humanor animal gastro-intestinal tissue, and derivatives (e.g., fragments)and analogs thereof, and nucleotide sequences coding for said proteinsand derivatives.

Preferably, the tissue through which transport is facilitated is of theduodenum, jejunum, ileum, ascending colon, transverse colon, descendingcolon, or pelvic colon. The tissue is most preferably epithelial cellslining the lumenal side of the GIT.

The proteins of the invention have use in facilitating transport ofactive agents from the lumenal side of the GIT into the systemic bloodsystem, and/or in targeting active agents to the GIT. Thus, for example,by binding (covalently or noncovalently) a protein of the invention toan orally administered drug, the drug can be targeted to specificreceptor sites or transport pathways which are known to operate in thehuman gastrointestinal tract, thus facilitating its absorption into thesystemic system.

The invention also relates to derivatives and analogs of the inventionwhich are functionally active, i.e., they are capable of displaying oneor more known functional activities associated with a full-lengthpeptide. Such functional activities include but are not limited toantigenicity (ability to bind or to compete with GIT transportreceptor-binding peptides for binding to an anti-GIT transport receptorantibody) and ability to bind or compete with full-length peptide forbinding to a GIT transport receptor.

The invention further relates to fragments of (and derivatives andanalogs thereof) GIT transport receptor-binding peptides which compriseone or more motifs of a GIT transport receptor-binding peptide.

Antibodies to GIT transport receptor-binding peptides and GIT transportreceptor-binding peptide derivatives and analogs are additionallyprovided.

Methods of production of the GIT transport receptor-binding peptides,derivatives, fragments and analogs, e.g., by recombinant means, are alsoprovided.

The present invention also relates to therapeutic methods,pharmaceutical compositions and formulations based on GIT transportreceptor-binding peptides. Formulations of the invention include but arenot limited to GIT transport receptor-binding peptides or motifs andderivatives (including fragments) thereof; antibodies thereto; andnucleic acids encoding the GIT transport receptor-binding peptides orderivatives associated with an active agent. Preferably, the activeagent is a drug or drug-containing nano- or microparticle.

The GIT transport-receptor binding proteins of the invention can also beused to determine levels of the GIT transport receptors in a sample bybinding thereto.

The GIT transport-receptor binding proteins can also be used to identifymolecules that bind thereto, by contacting candidate test moleculesunder conditions conducive to binding, and detecting any binding thatoccurs.

4. DESCRIPTION OF THE FIGURES

FIG. 1. FIG. 1 shows the human PEPT1 predicted amino acid sequencedetermined from the sequence of the cDNA clone coding for human PEPT1(SEQ ID NO:176) (Liang R. et al. J. Biol. Chem. 270(12):6456–6463(1995)), including the extracellular domain from amino acid 391 to 573(Fei et al., Nature 368:563 (1994)).

FIGS. 2A–2C. FIGS. 2A–2C show the DNA sequence of the cDNA coding forthe human intestinal peptide-associated transporter HPT1 and thecorresponding putative amino acid sequence (bases 1 to 3345;Medline:94204643) (SEQ ID NOS: 177 and 178, respectively).

FIGS. 3A–3B. FIGS. 3A–3B show the putative Human Sucrase-isomaltasecomplex(hSI) amino acid sequence determined from the sequence of thecDNA clone coding for human sucrase-isomaltase complex (SEQ ID NO:179)(Chantret I., et al., Biochem. J. 285(Pt 3):915–923 (1992).

FIGS. 4A–4B. FIGS. 4A–4B show the D2H nucleotide and deduced amino acidsequence for the human D2H transporter (SEQ ID NOS:180 and 181,respectively) (Wells, R. G. et al., J. Clin. Invest. 90:1959–1963(1993).

FIGS. 5A–5C. FIG. 5A is a schematic summary of the cloning of the DNAinsert present in gene III of the phages selected from the phage displaylibraries into the expression vector pGex-4T-2. The gene insert in geneIII of the phages was amplified by PCR using DNA primers which flank thegene insert and which contained recognition sequences for specificrestriction endonucleases at their extreme 5′ sides. Alternatively,specific primers which amplify specific regions of the DNA inserts ingene III of the phages, and which contained recognition sequences forspecific restriction endonucleases at their extreme 5′ sides, were usedin PCR amplification experiments. Following amplification of the geneinserts, the amplified PCR fragments were digested with the restrictionendonucleases Xho1 and Not1. Similarly the plasmid pGex-4T-2, whichcodes for the reporter protein glutathione S-transferase (GST), wasdigested with the restriction endonucleases Sal1 and Not1. The digestedPCR fragments were ligated into the digested plasmid pGex-4T-2 using T4DNA Ligase and the ligated products were transformed into competentEscherichia coli, with selection of transformants on agar platescontaining selection antibiotic. The selected clones were cultured, theplasmids were recovered and the in-frame sequence of the DNA insert inthe plasmids was confirmed by DNA sequencing. The correct clones weresubsequently used for expression of the GST-fusion proteins (SEQ IDNO:182); FIG. 5B shows the series of full-length P31 (designated P31)(SEQ ID NO:43) and truncated peptides derived from P31 (clones # 101,102, 103 and 119), (SEQ ID NOS:183, 184, 185, and 186, respectively)full-length PAX2 (designated PAX2) (SEQ ID NO:55) and truncated peptidesderived from PAX2 (clones # 104, 105, 106) (SEQ ID NOS:170, 187, and188, respectively) and full-length DCX8 (DCX8) (SEQ ID NO:23) and seriesof truncated peptides derived from DCX8 (clones # 107, 108, 109) (SEQ IDNOS:189, 190, and 191, respectively) that were expressed as fusionproteins to GST. The construction of these GST-fusion proteins is shownin FIG. 5A. FIG. 5C shows the series of full-length P31 (designated P31)(SEQ ID NO:43) and truncated peptides derived from P31 (clones # 103,110, 119, 111, and 112) (SEQ ID NOS:185, 192, 186, 194, and 195,respectively), full-length PAX2 (designated PAX2) (SEQ ID NO:55) andtruncated peptides derived from PAX2 (clones # 106, 113, 114, 115) (SEQID NOS:188, 196, 197, and 198, respectively) and full-length SNi10(designated SNi10) (SEQ ID NO:4) and series of truncated peptidesderived from SNi10 (clones # 116, 117, 118) (SEQ ID NOS:199, 200, and405, respectively) that were expressed as fusion proteins to GST. Theconstruction of these GST-fusion proteins is shown in FIG. 5A.(Underlining and bold in FIGS. 5A–5C are for orientation of thesequences.)

FIGS. 6A–6B. FIGS. 6A–6B show the binding of GST and GST-fusion proteinsto recombinant hSI and to fixed C2BBe1 fixed cells as detected by ELISAassays. FIG. 6A shows the binding of the control protein GST, which doesnot contain a fusion peptide, and the GST-fusion proteins from SNi10(designated GST-SNi10) and SNi34 (designated GST-SNi34) to recombinanthSI. FIG. 6B shows the binding of the control protein GST, which doesnot contain a fusion peptide, and the GST-fusion proteins from SNi10(designated GST-SNi10) and SNi34 (designated GST-SNi34) to fixed C2BBe1cells.

FIGS. 7A–7M. FIGS. 7A–7M show the binding of GST peptide and truncatedfusion proteins to fixed Caco-2 cells, fixed C2BBe1 cells, and fixedA431 cells or to recombinant GIT transport receptors D2H, HPT1, hPEPT1or to BSA using increasing concentrations (expressed as μg/ml on theX-axis) of the control GST protein and the GST-fusion proteins, asdetected by ELISA assays. FIG. 7A shows the binding of the controlprotein GST, which does not contain a fusion peptide, and the series ofGST-fusion proteins from P31 including the fusion to full-length P31peptide (designated P31) (SEQ ID NO:43) and clone # 101 (designatedP31,101, SEQ ID NO: 183), clone # 102 (designated P31, 102 SEQ ID NO:184) and clone # 103 (designated P31,103 SEQ ID NO: 185). FIG. 7B showsthe binding of the control protein GST, which does not contain a fusionpeptide, and the series of GST-fusion proteins from PAX2 including thefusion to full-length PAX2 peptide (designated PAX2) and clone # 104(designated PAX2,104), clone # 105 (designated PAX2, 105) and clone #106 (designated PAX2,106) (SEQ ID NOS:55, 170, 187, and 188,respectively). FIG. 7C shows the binding of the control protein GST,which does not contain a fusion peptide, and the series of GST-fusionproteins from DCX8 including the fusion to full-length DCX8 peptide(designated DCX8) and clone # 107 (designated DCX8,107), clone # 108(designated DCX8, 108) and clone # 109 (designated DCX8,109) (SEQ IDNOS:23, 189, 190, and 191, respectively). FIG. 7D shows the binding ofthe control protein GST, which does not contain a fusion peptide, andthe GST-fusion proteins from DCX8 (designated GST-DCX8) and DCX11(designated GST-DCX11) to recombinant D2H. FIG. 7E shows the binding ofthe control protein GST, which does not contain a fusion peptide, andthe GST-fusion proteins from DCX8 (designated GST-DCX8) and DCX11(designated GST-DCX11) to fixed C2BBe1 cells. FIG. 7F shows the bindingof the control protein GST, which does not contain a fusion peptide, andthe GST-fusion proteins from P31 (designated GST-P31) and 5PAX5(designated GST-5PAX5) to recombinant hPEPT1. FIG. 7G shows the bindingof the control protein GST, which does not contain a fusion peptide, andthe GST-fusion proteins from P31 (designated GST-P31) and 5PAX5(designated GST-5PAX5) to fixed C2BBe1 cells. FIG. 7H shows the bindingof the control protein GST, which does not contain a fusion peptide, andthe GST-fusion proteins from HAX42 (designated GST-HAX42) and PAX2(designated GST-PAX2) to recombinant HPT1. FIG. 7I shows the binding ofthe control protein GST, which does not contain a fusion peptide, andthe GST-fusion proteins from HAX42 (designated GST-HAX42) and PAX2(designated GST-PAX2) to fixed C2BBe1 cells. FIG. 7J shows the bindingof the control protein GST, which does not contain a fusion peptide, andthe GST-fusion proteins from P31 (designated GST-P31) and truncatedderivatives clone # 101 (designated GST-P31-101, SEQ ID NO: 183), clone# 102 (designated GST-P31-102, SEQ ID NO: 184), clone # 103 (designatedGST-P31-103, SEQ ID NO: 185) to either recombinant hPEPT1 or to BSA.FIG. 7K shows the binding of the control protein GST, which does notcontain a fusion peptide, and the GST-fusion proteins from P31(designated GST-P31) and truncated derivatives clone # 101 (designatedGST-P31-101, SEQ ID NO: 183), clone # 102 (designated GST-P31-102, SEQID NO: 184), clone # 103 (designated GST-P31-103, SEQ ID NO: 185) toeither fixed C2BBe1 cells or to fixed A431 cells. FIG. 7L shows thebinding of the control protein GST, which does not contain a fusionpeptide, and the GST-fusion proteins from PAX2 (designated GST-PAX2) andtruncated derivatives clone # 104 (designated GST-PAX2-104, SEQ ID NO:170), clone # 105 (designated GST-PAX2-105, SEQ ID NO: 187), clone # 106(designated GST-PAX2-106, SEQ ID NO: 188) to either recombinant hPEPT1or to BSA. FIG. 7M shows the binding of the control protein GST, whichdoes not contain a fusion peptide, and the GST-fusion proteins from PAX2(designated GST-PAX2) and truncated derivatives clone # 106 (designatedGST-PAX2-106, SEQ ID NO: 188)to either fixed Caco-2 cells or to fixedA431 cells.

FIGS. 8A–8D. FIG. 8 shows the transport of GST or GST-peptide fusionderivatives across polarized Caco-2 cells in an apical to basolateraldirection as a function of time (1–4 hours) as detected by ELISA assays.FIG. 8A shows the transport of either GST, the GST fusion to full-lengthP31 peptide (designated P31) (SEQ ID NO:43) and the GST clone derivativeclone # 103 (designated P31.103, SEQ ID NO: 185) across polarized Caco-2cells in an apical to basolateral as a function of time (in hours)following initial administration of the proteins to the apical medium ofpolarized Caco-2 cells. The line designated No Protein corresponds tocontrol assays in which buffer control was applied to the apical mediumof polarized Caco-2 cells followed by sampling of the basolateral mediumas a function of time (hours) and assay for GST by the ELISA assay. FIG.8B shows the transport of either GST, the GST fusion to full-length PAX2peptide (designated PAX2) and the GST clone derivative clone # 106(designated PAX2.106, SEQ ID NO: 188) across polarized Caco-2 cells inan apical to basolateral as a function of time (in hours) followinginitial administration of the proteins to the apical medium of polarizedCaco-2 cells. The line designated No Protein corresponds to controlassays in which buffer control was applied to the apical medium ofpolarized Caco-2 cells followed by sampling of the basolateral medium asa function of time (hours) and assay for GST by the ELISA assay. FIG. 8Cshows the transport of either GST, the GST fusion to full-length DCX8peptide (designated DCX8), and the GST clone derivatives clone # 107(designated DCX8.107) and clone # 109 (designated DCX8.109) acrosspolarized Caco-2 cells in an apical to basolateral as a function of time(in hours) following initial administration of the proteins to theapical medium of polarized Caco-2 cells. The line designated No Proteincorresponds to control assays in which buffer control was applied to theapical medium of polarized Caco-2 cells followed by sampling of thebasolateral medium as a function of time (hours) and assay for GST bythe ELISA assay. FIG. 8D shows the amount of the GST and GST-fusionproteins (GST fusions to P31, P31-103 (SEQ ID NO: 183), PAX2, PAX2.106(SEQ ID NO: 188), DCX8, DCX8-107, DCX8-109), used in the experimentsshown in panels A–C above, in the apical medium of the polarized Caco-2cells as detected by ELISA assay.

FIGS. 9A–9B. FIGS. 9A–9B show the inhibition of GST-P31 binding toC2BBe1 fixed cells with varying concentration of competitors whileholding the concentration of GST-P31 constant at 0.015 μM; the peptidecompetitors are ZElan024 (SEQ ID NO:288) which is the dansylated peptideversion of P31 (SEQ ID NO:43) and ZElan044 (SEQ ID NO:310), ZElan049(SEQ ID NO:315) and ZElan050 (SEQ ID NO:316) which are truncated,dansylated pieces of P31 (SEQ ID NO:43). Data is presented as O.D.versus peptide concentration (FIG. 9A) and as percent inhibition ofGST-P31 binding versus peptide concentration (FIG. 9B).

FIGS. 10A–10C. FIGS. 10A–10C present a compilation of the results ofcompetition ELISA studies of GST-P31, GST-PAX2, GST-SNi10 and GST-HAX42versus listed dansylated peptides on fixed C2BBe1 cells (“Z” denotesε-amino dansyl lysine). The pI of the dansylated peptides is alsoincluded. Estimated IC₅₀ values are in μM and where present, IC₅₀ rangesrefer to results from multiple assays. If the IC₅₀ value could not bedetermined, a “>” or “<” symbol is used. The GST/C2BBe1 column shows GSTprotein binding to fixed C2BBe1 cells.

FIGS. 11A–11B. FIG. 11A shows the transport of GST or GST-peptide fusionderivatives across polarized Caco-2 cells in an apical to basolateraldirection at 0, 0.5, 2 and 4 hours as detected by ELISA assays anddescribed elsewhere in the text in full detail. The proteins used in theassay included GST, GST-P31 fusion, GST-5PAX5 fusion, GST-DCX8 fusion,GST-DCX11 fusion, GST-PAX2 fusion, GST-HAX42 fusion, GST-SNi34 fusionand GST-SNi10 fusion. The column designated No protein refers to controlexperiments in which buffer was applied to the apical medium of thecells and ELISA assay was performed on the corresponding basolateralmedium of these cells at 0, 0.5, 2 and 4 hours post buffer addition.FIG. 11B shows the internalization of GST or GST-peptide fusionderivatives within polarized Caco-2 cells following administration ofthe GST or GST-fusion protein derivatives to the apical medium ofpolarized Caco-2 cells and subsequent recovery of the cells from thetranswells and detection of the GST or GST fusions within the recoveredcell lysates as detected by ELISA assays and as described elsewhere inthe text in full detail. The proteins used in the assay included GST,GST-P31 fusion, GST-5PAX5 fusion, GST-DCX8 fusion, GST-DCX11 fusion,GST-PAX2 fusion, GST-HAX42 fusion, GST-SNi34 fusion and GST-SNi10fusion. The column designated No protein refers to control experimentsin which buffer was applied to the apical medium of the cells and ELISAassay was performed on the corresponding cell lysates of these cells atthe end of the experiment.

FIG. 12. FIG. 12 shows the binding of GST and GST-fusion proteins tofixed Caco-2 cells, and the corresponding proteins following digestionwith the protease Thrombin which lysine). The pI of the dansylatedpeptides is also included. Estimated IC₅₀ values are in μM and wherepresent, IC₅₀ ranges refer to results from multiple assays. If the IC₅₀value could not be determined, a “>” or “<” symbol is used. TheGST/C2BBe1 column shows GST protein binding to fixed C2BBe1 cells.

FIGS. 11A–11B. FIG. 11A shows the transport of GST or GST-peptide fusionderivatives across polarized Caco-2 cells in an apical to basolateraldirection at 0, 0.5, 2 and 4 hours as detected by ELISA assays anddescribed elsewhere in the text in full detail. The proteins used in theassay included GST, GST-P31 fusion, GST-5PAX5 fusion, GST-DCX8 fusion,GST-DCX11 fusion, GST-PAX2 fusion, GST-HAX42 fusion, GST-SNi34 fusionand GST-SNi10 fusion. The column designated No protein refers to controlexperiments in which buffer was applied to the apical medium of thecells and ELISA assay was performed on the corresponding basolateralmedium of these cells at 0, 0.5, 2 and 4 hours post buffer addition.FIG. 11B shows the internalization of GST or GST-peptide fusionderivatives within polarized Caco-2 cells following administration ofthe GST or GST-fusion protein derivatives to the apical medium ofpolarized Caco-2 cells and subsequent recovery of the cells from thetranswells and detection of the GST or GST fusions within the recoveredcell lysates as detected by ELISA assays and as described elsewhere inthe text in full detail. The proteins used in the assay included GST,GST-P31 fusion, GST-5PAX5 fusion, GST-DCX8 fusion, GST-DCX11 fusion,GST-PAX2 fusion, GST-HAX42 fusion, GST-SNi34 fusion and GST-SNi10fusion. The column designated No protein refers to control experimentsin which buffer was applied to the apical medium of the cells and ELISAassay was performed on the corresponding cell lysates of these cells atthe end of the experiment.

FIG. 12. FIG. 12 shows the binding of GST and GST-fusion proteins tofixed Caco-2 cells, and the corresponding proteins following digestionwith the protease Thrombin which cleaves at a recognition site betweenthe GST portion and the fused peptide portion of the GST-fusion protein.The symbol “−” refers to proteins which were not digested with thrombinand the symbol “+” refers to proteins which were digested with thrombinprior to use in the binding assay. The binding of the proteins to thefixed Caco-2 cells was detected by ELISA assays.

FIGS. 13A–13B. FIGS. 13A–13B show binding of peptide-coatednanoparticles to fixed Caco-2 cells.

FIGS. 14A–14B. FIGS. 14A–14B show the binding of (A) dansylated peptideSNi10 to the purified hSI receptor and BSA and (B) dansylated peptidesand peptide-loaded insulin-containing PLGA particles to fixed C2BBe1cells. FIG. 14B depicts binding of dansylated peptides corresponding toP31 (SEQ ID NO:43), PAX2, HAX42, and SNi10 to fixed C2BBe1 cells, aswell as the insulin-containing PLGA particles adsorbed with each ofthese peptides. Data is presented with background subtracted.

FIGS. 15A–15B. FIG. 15 shows the binding of peptide-coated particles toA) S100 and B) P100 fractions harvested from Caco-2 cells. The dilutionseries 1:2–1:64 represents particle concentrations in the range0.0325–0.5 μg/well. Data is presented with background subtracted. Theparticles are identified as follows: 939, no peptide; 1635, scrambledPAX2; 1726, P31 D-Arg 16-mer (ZElan053, SEQ ID NO:319); 1756, HAX42;1757, PAX2; 1758, HAX42/PAX2.

FIGS. 16A–16B. FIG. 16 shows the binding of dansylated peptides to P100fractions harvested from Caco-2 cells. Peptides were assayed in therange 0.0032–2.5 μg/well. Data is presented with background subtracted.A) HAX42, P31 D-form (ZElan 053, (SEQ ID NO:319) and scrambled PAX2; B)PAX2, HAX42 and scrambled PAX2.

FIGS. 17A–17B. FIGS. 17A and 17B show (A) the systemic blood glucose and(B)insulin levels following intestinal administration of control (PBS);insulin solution; insulin particles; all 8 peptides mix particles andstudy group peptide-particles according to this invention (100 iuinsulin loading).

FIGS. 18A–18B. FIGS. 18A and 18B show the (A) systemic blood glucose and(B)insulin levels following intestinal administration of control (PBS);insulin solution; insulin particles and study group peptide-particlesaccording to this invention (300 iu insulin loading).

FIG. 19. FIG. 19 shows the enhanced plasma levels of leuprolide uponadministration of P31 (SEQ ID NO:43) and PAX2 coated nanoparticlesloaded with leuprolide relative to subcutaneous injection. Group 1 wasadministered leuprolide acetate (12.5 μg) subcutaneously. Group 2 wasadministered intraduodenally uncoated leuprolide acetate particles (600μg, 1.5 ml). Group 3 was intraduodenally administered leuprolide acetateparticles coated with PAX2 (600 μg; 1.5 ml). Group 4 was administeredintraduodenally leuprolide acetate particles coated with P31 (SEQ IDNO:43) (600 μg, 1.5 ml).

FIG. 20. FIG. 20 lists P31 (SEQ ID NO:43) known protein homologies.

FIGS. 21A–21C. FIGS. 21A–21C list DCX8 known protein homologies.

FIG. 22. FIG. 22 lists DAB10 known protein homologies.

FIG. 23. FIG. 23 shows the DNA sequence (SEQ ID NO:211) and thecorresponding amino acid sequence (SEQ ID NO:212) for glutathioneS-transferase (Smith and Johnson, 1988, Gene 7:31–40).

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to proteins (e.g., peptides) that bind toGIT transport receptors and nucleic acids that encode such proteins. Theinvention further relates to fragments and other derivatives of suchproteins. Nucleic acids encoding such fragments or derivatives are alsowithin the scope of the invention. The invention further relates tofragments (and derivatives and analogs thereof) of GIT transportreceptor-binding peptides which comprise one or more domains of the GITtransport receptor-binding peptides.

The invention also relates to derivatives of GIT transportreceptor-binding proteins and analogs of the invention which arefunctionally active, i.e., they are capable of displaying one or moreknown functional activities associated with a full-length GIT transportreceptor-binding peptide. Such functional activities include but are notlimited to ability to bind to a GIT transport receptor, antigenicity[ability to bind (or compete with peptides for binding) to an anti-GITtransport receptor-binding peptide antibody], immunogenicity (ability togenerate antibody which binds to GIT transport receptor-bindingpeptide), etc.

Production of the foregoing proteins and derivatives, by, e.g.,recombinant methods, is also provided.

Antibodies to GIT transport receptor-binding proteins, derivatives andanalogs, are additionally provided.

The present invention also relates to therapeutic and diagnostic methodsand compositions based on GIT transport receptor-binding proteins andnucleic acids.

The invention is illustrated by way of examples infra.

For clarity of disclosure, and not by way of limitation, the detaileddescription of the invention is divided into the subsections whichfollow.

5.1. GIT Transport Receptor-Binding Peptides, Derivatives and Analogs

The invention relates to peptides that bind GIT transport receptors andderivatives (including but not limited to fragments) and analogsthereof. In specific embodiments, of the present invention, suchpeptides that bind to GIT transport receptor include but are not limitedto those containing as primary amino acid sequences, all or part of theamino acid sequences substantially as depicted in Table 7 (SEQ IDNOS:1–55). Nucleic acids encoding such peptides, derivatives and peptideanalogs are also provided. In one embodiment, the GIT transportreceptor-binding peptides are encoded by the nucleic acids having thenucleotide sequences set forth in Table 8 infra (SEQ ID NOS:56–109).Proteins whose amino acid sequence comprise, or alternatively, consistof SEQ ID NOS:1–55 or a portion thereof that mediates binding to a GITtransport receptor are provided.

The production and use of derivatives and analogs related to GITtransport receptor-binding peptides are within the scope of the presentinvention. In a specific embodiment, the derivative or analog isfunctionally active, i.e., capable of exhibiting one or more functionalactivities associated with a full-length GIT transport receptor-bindingpeptide. For example, such derivatives or analogs which have the desiredimmunogenicity or antigenicity can be used, in immunoassays, forimmunization, etc. A specific embodiment relates to a GIT transportreceptor-binding peptide fragment that can be bound by an anti-GITtransport receptor-binding peptide antibody. In a preferred aspect, thederivatives or analogs have the ability to bind to a GIT transportreceptor. Derivatives or analogs of GIT transport receptor-bindingpeptides can be tested for the desired activity by procedures known inthe art, including binding to a GIT transport receptor domain or toCaco-2 cells, in vitro, or to intestinal tissue, in vivo. (See theExamples infra.)

In particular, derivatives can be made by altering GIT transportreceptor-binding peptide sequences by substitutions, additions ordeletions that provide for functionally equivalent molecules. Due to thedegeneracy of nucleotide coding sequences, other nucleotide sequenceswhich encode substantially the same amino acid sequence may be used inthe practice of the present invention. These include but are not limitedto nucleotide sequences which are altered by the substitution ofdifferent codons that encode a functionally equivalent amino acidresidue within the sequence, thus producing a silent change. Likewise,the GIT transport receptor-binding peptide derivatives of the inventioninclude, but are not limited to, those containing, as a primary aminoacid sequence, all or part of the amino acid sequence of a GIT transportreceptor-binding peptide including altered sequences in whichfunctionally equivalent amino acid residues are substituted for residueswithin the sequence resulting in a silent change. For example, one ormore amino acid residues within the sequence can be substituted byanother amino acid of a similar polarity which acts as a functionalequivalent, resulting in a silent alteration. Substitutes for an aminoacid within the sequence may be selected from other members of the classto which the amino acid belongs. For example, the nonpolar (hydrophobic)amino acids include alanine, leucine, isoleucine, valine, proline,phenylalanine, tryptophan and methionine. The polar neutral amino acidsinclude glycine, serine, threonine, cysteine, tyrosine, asparagine, andglutamine. The positively charged (basic) amino acids include arginine,lysine and histidine. The negatively charged (acidic) amino acidsinclude aspartic acid and glutamic acid.

In a specific embodiment of the invention, proteins consisting of or,alternatively, comprising all or a fragment of a GIT transportreceptor-binding peptide consisting of at least 5, 10, 15, 20, 25, 30 or35 (contiguous) amino acids of the full-length GIT transportreceptor-binding peptide are provided. In a specific embodiment, suchproteins are not more than 20, 30, 40, 50, or 75 amino acids in length.Derivatives or analogs of GIT transport receptor-binding peptidesinclude but are not limited to those molecules comprising regions thatare substantially homologous to GIT transport receptor-binding peptidesor fragments thereof (e.g., at least 50%, 60%, 70%, 80% or 90% identity)(e.g., over an identical size sequence or when compared to an alignedsequence in which the alignment is done by a computer homology programknown in the art) or whose encoding nucleic acid is capable ofhybridizing to a coding GIT transport receptor-binding peptide sequence,under stringent, moderately stringent, or nonstringent conditions.

In a specific embodiment, the GIT transport receptor-binding derivativesof the invention are not known proteins with homology to the GITtransport receptor-binding peptides of the invention or portionsthereof.

The GIT transport receptor-binding peptide derivatives and analogs ofthe invention can be produced by various methods known in the art. Themanipulations which result in their production can occur at the gene orprotein level. For example, the cloned GIT transport receptor-bindingpeptide gene sequence can be modified by any of numerous strategiesknown in the art (Maniatis, T., 1990, Molecular Cloning, A LaboratoryManual, 2d ed., Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y.). The sequence can be cleaved at appropriate sites with restrictionendonuclease(s), followed by further enzymatic modification if desired,isolated, and ligated in vitro. In the production of the gene encoding aderivative or analog of GIT transport receptor-binding peptides, careshould be taken to ensure that the modified gene remains within the sametranslational reading frame uninterrupted by translational stop signals,in the gene region where the desired GIT transport receptor-bindingpeptides activity is encoded.

Additionally, nucleic acid sequences encoding the GIT transportreceptor-binding peptides can be mutated in vitro or in vivo, to createand/or destroy translation, initiation, and/or termination sequences, orto create variations in coding regions and/or form new restrictionendonuclease sites or destroy preexisting ones, to facilitate further invitro modification. Any technique for mutagenesis known in the art canbe used, including but not limited to, chemical mutagenesis, in vitrosite-directed mutagenesis (Hutchinson, C., et al., 1978, J. Biol. Chem253:6551), use of TAB® linkers (Pharmacia), use of PCR primerscontaining mutation(s) for use in amplification, etc.

Manipulations of GIT transport receptor-binding peptide sequences mayalso be made at the protein level. Included within the scope of theinvention are GIT transport receptor-binding peptide fragments or otherderivatives or analogs which are differentially modified during or aftertranslation or chemical synthesis, e.g., by glycosylation, acetylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to an antibody molecule or othercellular ligand, etc. Any of numerous chemical modifications may becarried out by known techniques, including but not limited to specificchemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8protease, NaBH₄; acetylation, formylation, oxidation, reduction;metabolic synthesis in the presence of tunicamycin; etc. In a specificembodiment, the amino- and/or carboxy-termini are modified.

In addition, GIT transport receptor-binding peptides and analogs andderivatives thereof can be chemically synthesized. For example, apeptide corresponding to all or a portion of a GIT transportreceptor-binding peptide which comprises the desired domain or whichmediates the desired activity in vitro, can be synthesized by use of apeptide synthesizer. Furthermore, if desired, nonclassical amino acidsor chemical amino acid analogs can be introduced as a substitution oraddition into the GIT transport receptor-binding peptide sequence.Non-classical amino acids include but are not limited to the D-isomersof the common amino acids, α-amino isobutyric acid, 4-aminobutyric acid,Abu, 2-amino butyric acid, γ-Abu, ε-Ahx, 6-amino hexanoic acid, Aib,2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine,norvaline, hydroxyproline, sarcosine, 30 citrulline, cysteic acid,t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine,β-alanine, fluoro-amino acids, designer amino acids such as β-methylamino acids, Cα-methyl amino acids, Nα-methyl amino acids, and aminoacid analogs in general. Furthermore, the amino acid can be D(dextrorotary) or L (levorotary).

In a specific embodiment, the GIT transport receptor-binding peptidederivative is a chimeric, or fusion, peptide comprising a GIT transportreceptor-binding peptide or fragment thereof (preferably consisting ofat least a domain or motif of the GIT transport receptor-bindingpeptide, or at least 6, 10, 15, 20, 25, 30 or all amino acids of the GITtransport receptor-binding peptides or a binding portion thereof) joinedat its amino- or carboxy-terminus via a peptide bond to an amino acidsequence of a different peptide. In one embodiment, such a chimericpeptide is produced by recombinant expression of a nucleic acid encodingthe protein (comprising a transport receptor-coding sequence joinedin-frame to a coding sequence for a different protein). Such a chimericproduct can be made by ligating the appropriate nucleic acid sequencesencoding the desired amino acid sequences to each other by methods knownin the art, in the proper coding frame, and expressing the chimericproduct by methods commonly known in the art. Alternatively, such achimeric product may be made by protein synthetic techniques, e.g., byuse of a peptide synthesizer. Chimeric genes comprising portions of GITtransport receptor fused to any heterologous protein-encoding sequencesmay be constructed. A specific embodiment relates to a chimeric proteincomprising a fragment of GIT transport receptor-binding peptides of atleast six amino acids.

In another specific embodiment, the GIT transport receptor-bindingpeptide derivative is a molecule comprising a region of homology with aGIT transport receptor-binding peptide. By way of example, in variousembodiments, a first protein region can be considered “homologous” to asecond protein region when the amino acid sequence of the first regionis at least 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, or 95% identical,when compared to any sequence in the second region of an equal number ofamino acids as the number contained in the first region or when comparedto an aligned sequence of the second region that has been aligned by acomputer homology program known in the art. For example, a molecule cancomprise one or more regions homologous to a GIT transportreceptor-binding peptide domain (see infra) or a portion thereof.

The GIT transport receptor-binding proteins and derivatives thereof ofthe invention can be assayed for binding activity by suitable in vivo orin vitro assays, e.g., as described in the examples infra and/or as willbe known to the skilled artisan.

Other specific embodiments of derivatives and analogs are described inthe subsection below and examples sections infra.

5.2. Motifs/Derivatives of GIT Transport Receptor-Binding PeptidesContaining One or More Domains of the Protein

In a specific embodiment, the invention relates to GIT transportreceptor-binding peptide derivatives and analogs, in particular GITtransport receptor-binding peptide fragments and derivatives of suchfragments, that comprise, or alternatively consist of, one or moredomains of a GIT transport receptor-binding peptide. In particular,examples of such domains are identified in the examples infra.

5.3. Synthesis of Peptides

The peptides and derivatives of the present invention may be chemicallysynthesized or synthesized using recombinant DNA techniques.

5.3.1. Procedure For Solid Phase Synthesis

Peptides may be prepared chemically by methods that are known in theart. For example, in brief, solid phase peptide synthesis consists ofcoupling the carboxyl group of the C-terminal amino acid to a resin andsuccessively adding N-alpha protected amino acids. The protecting groupsmay be any known in the art. Before each new amino acid is added to thegrowing chain, the protecting group of the previous amino acid added tothe chain is removed. The coupling of amino acids to appropriate resinsis described by Rivier et al., U.S. Pat. No. 4,244,946. Such solid phasesyntheses have been described, for example, by Merrifield, 1964, J. Am.Chem. Soc. 85:2149; Vale et al., 1981, Science 213:1394–1397; Marki etal., 1981, J. Am. Chem. Soc. 103:3178 and in U.S. Pat. Nos. 4,305,872and 4,316,891. In a preferred aspect, an automated peptide synthesizeris employed.

By way of example but not limitation, peptides can be synthesized on anApplied Biosystems Inc. (“ABI”) model 431A automated peptide synthesizerusing the “Fastmoc” synthesis protocol supplied by ABI, which uses2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(“HBTU”) (R. Knorr et al., 1989, Tet. Lett., 30:1927) as coupling agent.Syntheses can be carried out on 0.25 mmol of commercially available4-(2′,4′-dimethoxyphenyl-(9-fluorenyl-methoxycarbonyl)-aminomethyl)-phenoxypolystyrene resin (“Rink resin” from Advanced ChemTech) (H. Rink, 1987,Tet. Lett. 28:3787). Fmoc amino acids (1 mmol) are coupled according tothe Fastmoc protocol. The following side chain protected Fmoc amino acidderivatives are used: FmocArg(Pmc)OH; FmocAsn(Mbh)OH; FmocAsp(^(t)Bu)OH;FmocCys(Acm)OH; FmocGlu(^(t)Bu)OH; FmocGln(Mbh)OH; FmocHis(Tr)OH;FmocLys(Boc)OH; FmocSer(^(t)Bu)OH; FmocThr(^(t)Bu)OH; FmocTyr(^(t)Bu)OH.[Abbreviations: Acm, acetamidomethyl; Boc, tert-butoxycarbonyl; ^(t)Bu,tert-butyl; Fmoc, 9-fluorenylmethoxycarbonyl; Mbh,4,4′-dimethoxybenzhydryl; Pmc, 2,2,5,7,8-pentamethylchroman-6-sulfonyl;Tr, trityl].

Synthesis is carried out using N-methylpyrrolidone (NMP) as solvent,with HBTU dissolved in N,N-dimethylformamide (DMF). Deprotection of theFmoc group is effected using approximately 20% piperidine in NMP. At theend of each synthesis the amount of peptide present is assayed byultraviolet spectroscopy. A sample of dry peptide resin (about 3–10 mg)is weighed, then 20% piperidine in DMA (10 ml) is added. After 30 minsonication, the UV (ultraviolet) absorbance of thedibenzofulvene-piperidine adduct (formed by cleavage of the N-terminalFmoc group) is recorded at 301 nm. Peptide substitution (in mmol g⁻¹)can be calculated according to the equation:substitution=

$\frac{A \times v}{7800 \times w} \times 1000$where A is the absorbance at 301 nm, v is the volume of 20% piperidinein DMA (in ml), 7800 is the extinction coefficient (in mol⁻¹dm³ cm⁻¹) ofthe dibenzofulvene-piperidine adduct, and w is the weight of thepeptide-resin sample (in mg).

Finally, the N-terminal Fmoc group is cleaved using 20% piperidine inDMA, then acetylated using acetic anhydride and pyridine in DMA. Thepeptide resin is thoroughly washed with DMA, CH₂Cl₂ and finally diethylether.

5.3.2. Cleavage and Deprotection

By way of example but not limitation, cleavage and deprotection can becarried out as follows: The air-dried peptide resin is treated withethylmethyl-sulfide (EtSMe), ethanedithiol (EDT), and thioanisole(PhSMe) for approximately 20 min. prior to addition of 95% aqueoustrifluoracetic acid (TFA). A total volume of approximately 50 ml ofthese reagents per gram of peptide-resin is used. The following ratio isused: TFA:EtSMe:EDT:PhSMe (10:0.5:0.5:0.5). The mixture is stirred for 3h at room temperature under an atmosphere of N₂. The mixture is 30filtered and the resin washed with TFA (2×3 ml). The combined filtrateis evaporated in vacuo, and anhydrous diethyl ether added to theyellow/orange residue. The resulting white precipitate is isolated byfiltration. See King et al., 1990, Int. J. Peptide Protein Res.36:255–266 regarding various cleavage methods.

5.3.3. Purification of the Peptides

Purification of the synthesized peptides can be carried out by standardmethods including chromatography (e.g., ion exchange, affinity, andsizing column chromatography, high performance liquid chromatography(HPLC)), centrifugation, differential solubility, or by any otherstandard technique.

5.3.4. Biological Peptide Libraries

Biological peptide libraries can be used to express and identifypeptides that bind to GIT transport receptors. According to this secondapproach, involving recombinant DNA techniques, peptides can, by way ofexample, be expressed in biological systems as either soluble fusionproteins or viral capsid proteins.

5.3.4.1. Methods to Identify Binders: Construction of Libraries

In a specific embodiment, the peptides of the invention thatspecifically bind to GIT transport receptors are identified by screeninga random peptide library by contacting the library with a ligandselected from among HPT1, hPEPT1, D2H, or hSI (or a molecule consistingessentially of an extracellular domain thereof or fragment of thedomain) to identify members of the library that specifically bind to theligand.

In a particular embodiment, a process to identify the peptides of thepresent method utilizes a library of recombinant vectors constructed bymethods well known in the art and comprises screening a library ofrecombinant vectors expressing inserted synthetic oligonucleotidesequences encoding extracellular GIT transport receptor domains, forexample, attached to an accessible surface structural protein of avector to isolate those members producing peptides that bind to HPT1,hPEPT1, D2H, or hSI. The nucleic acid sequence of the inserted syntheticoligonucleotides of the isolated vector is determined and the amino acidsequence encoded can be deduced to identify a binding domain that bindsthe ligand of choice (e.g., HPT1, hPEPT1, D2H, or hSI).

The present invention encompasses a method for identifying a peptidewhich binds to a ligand selected from among HPT1, hPEPT1, D2H, or hSIcomprising: screening a library of random peptides with the ligand (oran extracellular domain or fragment thereof) under conditions conduciveto ligand binding and isolating the peptide which binds to the ligand.Additionally, the methods of the invention further comprise determiningthe nucleotide sequence encoding the binding domain of the peptideidentified to deduce the amino acid sequence of the binding domain.

5.3.4.2. Preparation of Extracellular Domain Ligand

In a specific embodiment, molecules consisting essentially of anextracellular domain of the desired GIT transport receptor or a fragmentof an extracellular domain are used to screen a random peptide libraryfor binding thereto. Preferably, a nucleic acid encoding theextracellular domain is cloned and recombinantly expressed, and thedomain is then purified for use. The GIT transport receptor ispreferably selected from among HPT1, hPEPT1, D2H, or hSI.

5.3.4.3. Methods to Identify Binders: Screening Libraries

Once a suitable random peptide library has been constructed (orotherwise obtained), the library is screened to identify peptides havingbinding affinity for the GIT transport receptor, e.g., HPT1, hPEPT1,D2H, or hSI. In a preferred aspect, the library is a TSAR library (seeU.S. Pat. No. 5,498,538 dated Mar. 12, 1996 and PCT Publication WO94/18318 dated Aug. 18, 1994, both of which are incorporated byreference herein in their entireties). Screening the libraries can beaccomplished by any of a variety of methods known to those of skill inthe art. See, e.g., the following references, which disclose screeningof peptide libraries: Parmley and Smith, 1989, Adv. Exp. Med. Biol. 251:215–218; Scott and Smith, 1990, Science 249: 386–390; Fowlkes et al.,1992; BioTechniques 13: 422–427; Oldenburg et al., 1992, Proc. Natl.Acad. Sci. USA 89: 5393–5397; Yu et al., 1994, Cell 76: 933–945; Staudtet al., 1988, Science 241: 577–580; Bock et al., 1992, Nature 355:564–566; Tuerk et al., 1992, Proc. Natl. Acad. Sci. USA 89: 6988–6992;Ellington et al., 1992, Nature 355: 850–852; U.S. Pat. No. 5,096,815,U.S. Pat. No. 5,223,409, and U.S. Pat. No. 5,198,346, all to Ladner etal.; and Rebar and Pabo, 1993, Science 263: 671–673. See also PCTpublication WO 94/18318, dated Aug. 18, 1994.

One of ordinary skill in the art will recognize that, with suitablemodifications, the screening methods described below would be suitablefor a wide variety of biological expression libraries.

Once a library has been constructed or otherwise obtained, the libraryis screened to identify binding molecules having specific bindingaffinity for a ligand for a GIT transport receptor preferably selectedfrom among HPT1, hPEPT1, D2H, or hSI.

Screening the libraries can be accomplished by any of a variety ofmethods known to those of skill in the art. Exemplary screening methodsare described in Fowlkes et al., 1992, BioTechniques, 13:422–427 andinclude contacting the vectors with an immobilized target ligand andharvesting those vectors that bind to said ligand. Such useful screeningmethods, are designated “panning” methods. In panning methods useful toscreen the present libraries, the target ligand can be immobilized onplates, beads (such as magnetic beads), sepharose, beads used incolumns, etc. If desired, the immobilized target ligand can be “tagged”,e.g., using labels such as biotin, fluoroscein isothiocyanate,rhodamine, etc. e.g. for FACS sorting. Panning is also disclosed inParmley, S. F. and Smith, G. P., 1988, Gene 73: 305–318.

In a particular embodiment of the invention, the library can be screenedwith a recombinant receptor domain. In another embodiment, the librarycan be screened successively with receptor domains and then on CaCO-2cells.

For screening of the peptide libraries in vitro, the solventrequirements involved in screening are not limited to aqueous solvents;thus, nonphysiological binding interactions and conditions differentfrom those found in vivo can be exploited.

Screening a library can be achieved using a method comprising a first“enrichment” step and a second filter lift as follows. The followingdescription is given by way of example, not limitation.

Binders from an expressed library (e.g., in phage) capable of binding toa given ligand (“positives”) are initially enriched by one or two cyclesof panning or affinity chromatography. A microtiter well is passivelycoated with the ligand (e.g., about 10 μg in 100 μl). The well is thenblocked with a solution of BSA to prevent non-specific adherence of thephage of the library to the plastic surface. For example, about 10¹¹phage particles expressing peptides are then added to the well andincubated for several hours. Unbound phage are removed by repeatedwashing of the plate, and specifically bound phage are eluted using anacidic glycine-HCl solution or other elution buffer. The eluted phagesolution is neutralized with alkali, and amplified, e.g., by infectionof E. coli and plating on large petri dishes containing Luria broth (LB)in agar. Amplified cultures expressing the binding peptides are thentitered and the process repeated. Alternatively, the ligand can becovalently coupled to agarose or acrylamide beads using commerciallyavailable activated bead reagents. The phage solution is then simplypassed over a small column containing the coupled bead matrix which isthen washed extensively and eluted with acid or other eluant. In eithercase, the goal is to enrich the positives to a frequency of about>1/10⁵.

Following enrichment, a filter lift assay is conducted. For example,when specific binders are expressed in phage, approximately 1–2×10⁵phage are added to 500 μl of log phase E. coli and plated on a largeLuria Broth-agarose plate with 0.7% agarose in broth. The agarose isallowed to solidify, and a nitrocellulose filter (e.g., 0.45μ) is placedon the agarose surface. A series of registration marks is made with asterile needle to allow re-alignment of the filter and plate followingdevelopment as described below. Phage plaques are allowed to develop byovernight incubation at 37° C. (the presence of the filter does notinhibit this process). The filter is then removed from the plate withphage from each individual plaque adhered in situ. The filter is thenexposed to a solution of BSA or other blocking agent for 1–2 hours toprevent non-specific binding of the ligand (or “probe”).

The probe itself is labeled, for example, either by biotinylation (usingcommercial NHS-biotin) or direct enzyme labeling, e.g., with horseradish peroxidase or alkaline phosphatase. Probes labeled in this mannerare indefinitely stable and can be re-used several times. The blockedfilter is exposed to a solution of probe for several hours to allow theprobe to bind in situ to any phage on the filter displaying a peptidewith significant affinity to the probe. The filter is then washed toremove unbound probe, and then developed by exposure to enzyme substratesolution (in the case of directly labeled probe) or further exposed to asolution of enzyme-labeled avidin (in the case of biotinylated probe).Positive phage plaques are identified by localized deposition of coloredenzymatic cleavage product on the filter which corresponds to plaques onthe original plate. The developed filter is simply realigned with theplate using the registration marks, and the “positive” plaques are coredfrom the agarose to recover the phage. Because of the high density ofplaques on the original plate, it may be difficult to isolate a singleplaque from the plate on the first pass. Accordingly, phage recoveredfrom the initial core can be re-plated at low density and the processcan be repeated to allow isolation of individual plaques and hencesingle clones of phage.

Successful screening experiments are optimally conducted using 3 roundsof serial screening. The recovered cells are then plated at a lowdensity to yield isolated colonies for individual analysis. Theindividual colonies are selected and used to inoculate LB culture mediumcontaining ampicillin. After overnight culture at 37° C., the culturesare then spun down by centrifugation. Individual cell aliquots are thenretested for binding to the target ligand attached to the beads. Bindingto other beads having attached thereto a non-relevant ligand, can beused as a negative control.

One aspect of screening the libraries is that of elution. The followingdiscussion is applicable to any system where the random peptide isexpressed on a surface fusion molecule. It is conceivable that theconditions that disrupt the peptide-target interactions during recoveryof the phage are specific for every given peptide sequence from aplurality of proteins expressed on phage. For example, certaininteractions may be disrupted by acid pH but not by basic pH, and viceversa. Thus, it may be desirable to test a variety of elution conditions(including but not limited to pH 2–3, pH 12–13, excess target incompetition, detergents, mild protein denaturants, urea, varyingtemperature, light, presence or absence of metal ions, chelators, etc.)and compare the primary structures of the binding proteins expressed onthe phage recovered for each set of conditions to determine theappropriate elution conditions for each ligand/binding proteincombination. Some of these elution conditions may be incompatible withphage infection because they are bactericidal and will need to beremoved by dialysis (i.e., dialysis bag, Centricon/Amiconmicroconcentrators).

In a preferred embodiment, a phage display library of random peptides isscreened to select phage expressing peptides that bind to a GITtransport receptor. Preferably, a first step is to isolate a preselectedphage library. The “preselected phage library” is a library consistingof a subpopulation of a phage display library. This subpopulation can beformed by initially screening against either a target GIT transportreceptor (or domain thereof) so as to permit the selection of asubpopulation of phages which specifically bind to the receptor.Alternatively, the subpopulation can be formed by screening against atarget cell or cell type or tissue type or tissue barrier of thegastro-intestinal tract, so as to permit the selection of asubpopulation of phages which either bind specifically to the targetcell or target cell type or target tissue or target tissue barrier, orwhich binds to and/or is transported across (or between) the target cellor target cell type or target tissue or target tissue barrier either insitu or in vivo. This preselected phage library or subpopulation ofselected phages can also be rescreened against the target GIT transportreceptor, permitting the further selection of a subpopulation of phageswhich bind to the GIT transport receptor or target cell or target celltype or target tissue or target tissue barrier or which bind to and/oris transported across the target cell, target tissue or target tissuebarrier either in situ or in vivo. Such rescreening can be repeated fromzero to 30 times with each successive “pre-selected phage library”generating additional pre-selected phage libraries.

In a preferred embodiment, a preselected phage library binding a ligandthat is a GIT transport receptor preferably selected from among HPT1,hPEPT1, D2H, or hSI is obtained by an in vitro screening step asdescribed above, and then the phage are optionally further characterizedusing in vitro assays consisting of binding phage directly to thereceptor domain of interest or, alternatively, to Caco-2 cells or usingin vivo assays. In another preferred embodiment, in vivo assays are usedthat measure uptake of phage by intestinal tissue or, alternatively,through the GIT. In alternative embodiments, such further in vitro or invivo assays can be used as the initial screening step.

In vivo assays that may be used are described in the examples infra.

5.4. Generation of Antibodies to GIT Transport Receptor-Binding Peptidesand Derivatives Thereof

According to the invention, a GIT transport receptor-binding peptide,fragments or other derivatives, or analogs thereof, may be used as animmunogen to generate antibodies which immunospecifically bind such animmunogen. Such antibodies include but are not limited to polyclonal,monoclonal, chimeric, single chain, Fab fragments, and an Fab expressionlibrary.

Various procedures known in the art may be used for the production ofpolyclonal antibodies to a GIT transport receptor-binding peptide orderivative or analog. For the production of antibody, various hostanimals can be immunized by injection with the native GIT transportreceptor-binding peptides, or a synthetic version, or derivative (e.g.,fragment) thereof, including but not limited to rabbits, mice, rats,fowl, etc. Various adjuvants may be used to increase the immunologicalresponse, depending on the host species, including but not limited toFreund's (complete and incomplete), mineral gels such as aluminumhydroxide, surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanins, dinitrophenol, and potentially useful human adjuvants suchas BCG (bacille Calmette-Guerin) and corynebacterium parvum.

For preparation of monoclonal antibodies directed toward a GIT transportreceptor-binding peptide or analog thereof, any technique which providesfor the production of antibody molecules by continuous cell lines inculture may be used. For example, the hybridoma technique originallydeveloped by Kohler and Milstein (1975, Nature 256:495–497), as well asthe trioma technique, the human B-cell hybridoma technique (Kozbor etal., 1983, Immunology Today 4:72), and the EBV-hybridoma technique toproduce human monoclonal antibodies (Cole et al., 1985, in MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77–96). In anadditional embodiment of the invention, monoclonal antibodies can beproduced in germ-free animals utilizing recent technology(PCT/US90/02545). According to the invention, human antibodies may beused and can be obtained by using human hybridomas (Cote et al., 1983,Proc. Natl. Acad. Sci. U.S.A. 80:2026–2030) or by transforming human Bcells with EBV virus in vitro (Cole et al., 1985, in MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, pp. 77–96). According tothe invention, techniques developed for the production of “chimericantibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci. U.S.A.81:6851–6855; Neuberger et al., 1984, Nature 312:604–608; Takeda et al.,1985, Nature 314:452–454) by splicing the genes from a mouse antibodymolecule specific for GIT transport receptor-binding peptides togetherwith genes from a human antibody molecule of appropriate biologicalactivity can be used.

According to the invention, techniques described for the production ofsingle chain antibodies (U.S. Pat. No. 4,946,778) can be adapted toproduce GIT transport receptor-binding peptide-specific single chainantibodies. An additional embodiment of the invention utilizes thetechniques described for the construction of Fab expression libraries(Huse et al., 1989, Science 246:1275–1281) to allow rapid and easyidentification of monoclonal Fab fragments with the desired specificityfor GIT transport receptor-binding peptides, derivatives, or analogs.

Antibody fragments which contain the idiotype of the molecule can begenerated by known techniques. For example, such fragments include butare not limited to: the F(ab′)₂ fragment which can be produced by pepsindigestion of the antibody molecule; the Fab′ fragments which can begenerated by reducing the disulfide bridges of the F(ab′)₂ fragment, theFab fragments which can be generated by treating the antibody moleculewith papain and a reducing agent, and Fv fragments.

In the production of antibodies, screening for the desired antibody canbe accomplished by techniques known in the art, e.g. ELISA(enzyme-linked immunosorbent assay). For example, to select antibodieswhich recognize a specific domain of a GIT transport receptor-bindingpeptide, one may assay generated hybridomas for a product which binds toa GIT transport receptor-binding peptide fragment containing such adomain.

Antibodies specific to a domain of a GIT transport receptor-bindingpeptide are also provided.

The foregoing antibodies can be used in methods known in the artrelating to the localization and activity of the GIT transportreceptor-binding peptide sequences of the invention, e.g., for imagingthese peptides after in vivo administration (e.g., to monitor treatmentefficacy), measuring levels thereof in appropriate physiologicalsamples, in diagnostic methods, etc. For instance, antibodies orantibody fragments specific to a domain of a GIT transportreceptor-binding peptide or to a derivative of a peptide, such as adansyl group or some other epitope introduced into the peptide, can beused to 1) identify the presence of the peptide on a nanoparticle orother substrate; 2) quantify the amount of peptide on the nanoparticle;3)measure the level of the peptide in appropriate physiological samples;4) perform immunohistology on tissue samples; 5) image the peptide afterin vivo administration; 6) purify the peptide from a mixture using animmunoaffinity column or 7) bind or fix the peptide to the surface ofnanoparticle. This last use envisions attaching the antibody (orfragment of the antibody) to the surface of drug-loaded nanoparticles orother substrate and then incubating this conjugate with the peptide.This procedure results in binding of the peptide in a certain fixedorientation, resulting in a particle that contains the peptide bound tothe antibody in such a way that the peptide is fully active.

Abtides (or Antigen binding peptides) specific to a domain of a GITtransport receptor-binding peptide or to a derivative of a peptide, suchas a dansyl group or some other epitope introduced into the peptide, canbe used for the same seven purposes identified above for antibodies.

5.5. Assays of GIT Transport Receptor-Binding Peptides, Derivatives andAnalogs

The functional activity of GIT transport receptor-binding peptides,derivatives and analogs can be assayed by various methods.

In a preferred embodiment, in which binding to a GIT transport receptoris being assayed, the binding can be assayed by in vivo or in vitroassays such as described in the examples infra, or by other means thatare known in the art.

In another embodiment, where one is assaying for the ability to bind orcompete with full-length GIT transport receptor-binding peptide forbinding to anti-GIT transport receptor-binding peptide antibody, variousimmunoassays known in the art can be used, including but not limited tocompetitive and non-competitive assay systems using techniques such asradioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich”immunoassays, immunoradiometric assays, gel diffusion precipitinreactions, immunodiffusion assays, in situ immunoassays (using colloidalgold, enzyme or radioisotope labels, for example), western blots,precipitation reactions, agglutination assays (e.g., gel agglutinationassays, hemagglutination assays), complement fixation assays,immunofluorescence assays, protein A assays, and immunoelectrophoresisassays, etc. In one embodiment, antibody binding is detected bydetecting a label on the primary antibody. In another embodiment, theprimary antibody is detected by detecting binding of a secondaryantibody or reagent to the primary antibody. In a further embodiment,the secondary antibody is labelled. Many means are known in the art fordetecting binding in an immunoassay and are within the scope of thepresent invention.

Other methods will be known to the skilled artisan and are within thescope of the invention.

5.6. Uses

The invention provides compositions comprising the GIT transportreceptor-binding proteins of the invention bound to a materialcomprising an active agent. Such compositions have use in targeting theactive agent to the GIT and/or in facilitating transfer through thelumen of the GIT into the systemic circulation. Where the active agentis an imaging agent, such compositions can be administered in vivo toimage the GIT (or particular transport receptors thereof). Other activeagents include but are not limited to: any drug or antigen or any drug-or antigen-loaded or drug- or antigen-encapsulated nanoparticle,microparticle, liposome, or micellar formulation capable of eliciting abiological response in a human or animal. Examples of drug- orantigen-loaded or drug- or antigen-encapsulated formulations includethose in which the active agent is encapsulated or loaded into nano- ormicroparticles, such as biodegradable nano- or microparticles, and whichhave the GIT transport receptor-binding protein or derivative or analogadsorbed, coated or covalently bound, such as directly linked or linkedvia a linking moiety, onto the surface of the nano- or microparticle.Additionally, the protein, derivative or analog can form the nano- ormicroparticle itself or the protein, derivative or analog can becovalently attached to the polymer or polymers used in the production ofthe biodegradable nano- or microparticles or drug-loaded ordrug-encapsulated nano- or microparticles or the peptide can be directlyconjugated to the active agent. Such conjugations to active agentsinclude fusion proteins in which a DNA sequence coding for the peptideis fused in-frame to the gene or cDNA coding for a therapeutic peptideor protein such that the modified gene codes for a recombinant fusionprotein.

In a preferred embodiment, the invention provides for treatment ofvarious diseases and disorders by administration of a therapeuticcompound (termed herein “Therapeutic”). Such “Therapeutics” include butare not limited to: GIT transport receptor-binding proteins, and analogsand derivatives (including fragments) thereof (e.g., as describedhereinabove) that bind to GIT transport receptors, bound to an activeagent of value in the treatment or prevention of a disease or disorder(preferably a mammalian, most preferably human, disease or disorder).Therapeutics also include but are not limited to nucleic acids encodingthe GIT transport receptor-binding proteins, analogs, or derivativesbound to such a therapeutic or prophylactic active agent. The activeagent is preferably a drug.

Any drug known in the art may be used, depending upon the disease ordisorder to be treated or prevented, and the type of subject to which itis to be administered. As used herein, the term “drug” includes, withoutlimitation, any pharmaceutically active agent. Representative drugsinclude, but are not limited to, peptides or proteins, hormones,analgesics, anti-migraine agents, anti-coagulant agents, anti-emeticagents, cardiovascular agents, anti-hypertensive agents, narcoticantagonists, chelating agents, anti-anginal agents, chemotherapy agents,sedatives, anti-neoplastics, prostaglandins, and antidiuretic agents.Typical drugs include peptides, proteins or hormones such as insulin,calcitonin, calcitonin gene regulating protein, atrial natriureticprotein, colony stimulating factor, betaseron, erythropoietin (EPO),interferons such as α, β or γ interferon, somatropin, somatotropin,somatostatin, insulin-like growth factor (somatomedins), luteinizinghormone releasing hormone (LHRH), tissue plasminogen activator (TPA),growth hormone releasing hormone (GHRH), oxytocin, estradiol, growthhormones, leuprolide acetate, factor VIII, interleukins such asinterleukin-2, and analogs thereof; analgesics such as fentanyl,sufentanil, butorphanol, buprenorphine, levorphanol, morphine,hydromorphone, hydocodone, oxymorphone, methadone, lidocaine,bupivacaine, diclofenac, naproxen, paverin, and analogs thereof;anti-migraine agents such as heparin, hirudin, and analogs thereof;anti-coagulant agents such as scopolamine, ondansetron, domperidone,etoclopramide, and analogs thereof; cardiovascular agents,anti-hypertensive agents and vasodilators such as diltiazem, clonidine,nifedipine, verapamil, isosorbide-5-mononitrate, organic nitrates,agents used in treatment of heart disorders and analogs thereof;sedatives such as benzodiazeines, phenothiozines and analogs thereof;narcotic antagonists such as naltrexone, naloxone and analogs thereof;chelating agents such as deferoxamine and analogs thereof; anti-diureticagents such as desmopressin, vasopressin and analogs thereof;anti-anginal agents such as nitroglycerine and analogs thereof;anti-neoplastics such as 5-fluorouracil, bleomycin and analogs thereof;prostaglandins and analogs thereof; and chemotherapy agents such asvincristine and analogs thereof. Representative drugs also include butare not limited to antisense oligonucleotides, genes, gene correctinghybrid oligonucleotides, ribozymes, aptameric oligonucleotides,triple-helix forming oligonucleotides, inhibitors of signal transductionpathways, tyrosine kinase inhibitors and DNA modifying agents. Drugsthat can be used also include, without limitation, systems containinggene therapeutics, including viral systems for therapeutic gene deliverysuch as adenovirus, adeno-associated virus, retroviruses, herpes simplexvirus, sindbus virus, liposomes, cationic lipids, dendrimers, andenzymes. For instance, gene delivery viruses can be modified such thatthey express the targeting peptide on the surface so as to permittargeted gene delivery.

In a preferred embodiment, a Therapeutic is therapeutically orprophylactically administered to a human patient.

Additional descriptions and sources of Therapeutics that can be usedaccording to the invention are found in various Sections herein.

5.7. Therapeutic/Prophylactic Administration, Compositions andFormulations

The invention provides methods of treatment (and prophylaxis) byadministration to a subject of an effective amount of a Therapeutic ofthe invention. In a preferred aspect, the Therapeutic is substantiallypurified. The subject is preferably an animal, including but not limitedto animals such as cows, pigs, horses, chickens, cats, dogs, etc., andis preferably a mammal, and most preferably a human.

As will be clear, any disease or disorder of interest amenable totherapy or prophylaxis by providing a drug in vivo systemically or bytargeting a drug in vivo to the GIT (by linkage to a GITtransport-receptor binding protein, derivative or analog of theinvention) can be treated or prevented by administration of aTherapeutic of the invention. Such diseases may include but are notlimited to hypertension, diabetes, osteoporosis, hemophilia, anemia,cancer, migraine, and angina pectoris, to name but a few.

Any route of administration known in the art may be used, including butnot limited to oral, nasal, topical, intravenous, intraperitoneal,intradermal, mucosal, intrathecal, intramuscular, etc. Preferably,administration is oral; in such an embodiment the GIT-transport bindingprotein, derivative or analog of the invention acts advantageously tofacilitate transport of the therapeutic active agent through the lumenof the GIT into the systemic circulation.

The present invention also provides therapeuticcompositions/formulations. In a specific embodiment of the invention, aGIT transport receptor-binding peptide or motif of interest isassociated with a therapeutically or prophylactically active agent,preferably a drug or drug-containing nano- or microparticle. Morepreferably, the active agent is a drug encapsulating or drug loadednano- or microparticle, such as a biodegradable nano- or microparticle,in which the peptide is physically adsorbed or coated or covalentlybonded, such as directly linked or linked via a linking moiety, onto thesurface of the nano- or microparticle. Alternatively, the peptide canform the nano- or microparticle itself or can be directly conjugated tothe active agent. Such conjugations include fusion proteins in which aDNA sequence coding for the peptide is fused in-frame to the gene orcDNA coding for a therapeutic peptide or protein, such that the modifiedgene codes for a recombinant fusion protein in which the “targeting”peptide is fused to the therapeutic peptide or protein and where the“targeting” peptide increases the absorption of the fusion protein fromthe GIT. Preferably the particles range in size from 200–600 nm.

Thus, in a specific embodiment, a GIT transport-binding protein is boundto a slow-release (controlled release) device containing a drug. In aspecific embodiment, polymeric materials can be used (see MedicalApplications of Controlled Release, Langer and Wise (eds.), CRC Pres.,Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug ProductDesign and Performance, Smolen and Ball (eds.), Wiley, N.Y. (1984);Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983);see also Levy et al., Science 228:190 (1985); During et al., Ann.Neurol. 25:351 (1989); Howard et al., J. Neurosurg. 71:105 (1989)).

The present invention also provides pharmaceutical compositions. Suchcompositions comprise a therapeutically effective amount of aTherapeutic, and a pharmaceutically acceptable carrier. In a specificembodiment, the term “pharmaceutically acceptable” means approved by aregulatory agency of the Federal or a state government or listed in theU.S. Pharmacopeia or other generally recognized pharmacopeia for use inanimals, and more particularly in humans. The term “carrier” refers to adiluent, adjuvant, excipient, or vehicle with which the therapeutic isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Water is a preferred carrier when the pharmaceuticalcomposition is administered intravenously. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. Suitable pharmaceuticalexcipients include starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene, glycol,water, ethanol and the like. The composition, if desired, can alsocontain minor amounts of wetting or emulsifying agents, or pH bufferingagents. These compositions can take the form of solutions, suspensions,emulsion, tablets, pills, capsules, powders, sustained-releaseformulations and the like. The composition can be formulated as asuppository, with traditional binders and carriers such astriglycerides. Oral formulations can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin. Such compositions will containa therapeutically effective amount of the Therapeutic, preferably inpurified form, together with a suitable amount of carrier so as toprovide the form for proper administration to the patient.

The Therapeutics of the invention can be formulated as neutral or saltforms. Pharmaceutically acceptable salts include those formed with freeamino groups such as those derived from hydrochloric, phosphoric,acetic, oxalic, tartaric acids, etc., and those formed with freecarboxyl groups such as those derived from sodium, potassium, ammonium,calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

The amount of the Therapeutic of the invention which will be effectivein the treatment of a particular disorder or condition will depend onthe nature of the disorder or condition, and can be determined bystandard clinical techniques. In addition, in vitro assays mayoptionally be employed to help identify optimal dosage ranges. Theprecise dose to be employed in the formulation will also depend on theroute of administration, and the seriousness of the disease or disorder,and should be decided according to the judgment of the practitioner andeach patient's circumstances.

6. EXAMPLES

6.1. Selection of GIT Receptor Targets

The HPT1, hPEPT1, D2H, and hSI receptors were selected for cloning asGIT receptor targets based on several criteria, including: (1)expression on surface of epithelial cells in gastro-intestinal tract(GIT); (2) expression along the length of small intestine (HPT1, hPEPT1,D2H); (3) expression locally at high concentration (hSI); (4) largeputative extracellular domains facing into the lumen of the GIT; and (5)extracellular domains that permit easy access and bioadhesion bytargeting particles.

The four recombinant receptor sites screened with the peptide librariesadditionally have the following characteristics:

Receptor Characteristics D2H Transport of neutral/basic amino acids; atransport activating protein for a range of amino acid translocases hSIMetabolism of sucrose and other sugars; represents 9% of brush bordermembrane protein in Jejunum HPT1 di/tri peptide transporter orfacilitator of peptide transport hPEPT1 di/tri peptide transporterFIGS. 1–4 (SEQ ID NOS:176, 178, 179, and 181, respectively) show thepredicted amino acid sequences for hPEPT1, HPT1, hSI and D2H,respectively.

6.2. Cloning of Extracellular Domain of Selected Receptor Site

The following receptor domains were cloned and expressed as His-tagfusion proteins by standard techniques:

Domain Receptor (amino acid residues) hPEPT1^(a) 391–571 HPT1^(b) 29–273 hSI^(c) 272–667 D2H^(d) 387–685 ^(a)Liang et al., 1995, J. Biol.Chem. 270: 6456–6463 ^(b)Dantzig et al., 1994, Association of IntestinalPeptide Transport with a Protein Related to the Cadherin Superfamily^(c)Chantret et al., Biochem. J. 285: 915–923 ^(d)Bertran et al., J.Biol. Chem. 268: 14842–14949

The receptor proteins were expressed as His-tag fusion proteins andaffinity purified under denaturing conditions, using urea or guanidineHCl, utilizing the pET His-tag metal chelate affinity for Ni-NTA Agarose(Hochuli, E., Purification of recombinant proteins with metal chelateadsorbent, Genetic Engineering, Principals and Methods (J. K. Setlow,ed.), Plenum Press, N.Y., Vol. 12 (1990), pp. 87–98).

6.3. Phage Libraries

Three phage DC8, D38, and DC43 libraries expressing N-terminal pIIIfusions in M13 were used to identify peptides that bind to the GITreceptors. The D38 and DC43 libraries which are composed of 37 and 43random amino acid domains, respectively, have been described previously(McConnell et al., 1995, Molecular Diversity, 1:165–176). The DC8library is similar to the other two except that the random insert is 8amino acids long flanked on each side by a cysteine residue (i.e.,CX₈C).

6.4. Biopanning

Three rounds of biopanning on the GIT receptors were performed generallyby standard methods (McConnell et al., 1995, Molecular Diversity,1:165–176), using a mixture of the DC8 (1×10¹⁰ pfu), D38 and DC43(1×10¹¹ pfu) phage libraries. After each round of panning the percentageof phage recovered was determined. Following the first two rounds ofpanning, the eluted phage were amplified overnight. Phage from the thirdpan were plated out and 100 plaques were picked, amplified overnight andscreened in an ELISA assay for binding to the relevant receptor and BSA.After data analysis, phage clones were identified which had highabsorbance in the ELISA assay and/or a good ratio of binding to targetcompared to binding to BSA. The Insulin Degrading Enzyme (IDE) andrecombinant human tissue factor (hTF) were used as irrelevant controls.Several variations of the standard panning technique, discussed below,were used. Selection or panning methods followed one of two strategies.The first strategy involved panning the mixed libraries on the specificGIT receptor adsorbed to a solid surface. The second strategy panned thelibraries twice against the GIT receptor and then against Caco-2 cells(Peterson and Mooseker, 1992, J. Cell Science 102:581–600), Selectionmethods are reflected in the clone nomenclature as described below:

S designates the clone was identified by binding to the hS1 receptordomain.

D designates the clone was identified by binding to the D2H receptordomain.

P designates the clone was identified by binding to the PEPT1 receptordomain.

H designates the clone was identified by binding to the HPT-1 receptordomain.

Phage designated Ni are from a solid phase band GIT receptor pan thatused the standard procedure with the addition of Ni-NTA Agarose (Qiagen,Chatsworth, Calif.). Receptor coated plates were blocked with 0.5%BSA/PBS containing 160 μl Ni-NTA agarose and libraries were panned inthe presence of 50 μl Ni-NTA agarose. The receptor proteins wereexpressed as His-tag fusions. The His-tag has a high affinity for Ni-NTAAgarose. Blocking the plate and panning in the presence of Ni-NTAagarose minimized phage binding to the His-tag portion of therecombinant receptor.

Phage with the designation AX were eluted with acid and Factor Xa. Phagewere first eluted by standard acid elution then Factor Xa (New EnglandBiolabs, Beverly, Mass.: 1 μg protease in 300 μl of 20 mM Tris-HCL, 100mM NaCl, 2 mM CaCl₂) was added to the panning plate and incubated 2hours. Phage from both elution methods were pooled together then plated.

Phage with the designation AB were eluted with acid and base. Phage wereeluted first by standard acid elution then 100 mM triethylamine pH 12.1was added to the panning plate for 10 minutes. Phage from both elutionmethods were pooled together then plated.

C designates panning on receptor followed by Caco-2 cells. First andsecond round pans were performed on the receptor and the third round panwas on snapwells of Caco-2 cells. DCX11, DCX8 and DCX33 were identifiedby two pans on D2H receptor, third pan on Caco-2 cells. The third roundFactor Xa eluate from the Caco-2 cells was screened by ELISA on D2H, BSAand fixed Caco-2 cells. For HCA3 the first two rounds of panning wereperformed on the HPT-1 receptor and the third pan was on monolayerscultured on snapwells of Caco-2 cells.

Phage designated 5PAX were carried through five rounds of panning afterwhich a number of phage were sequenced prior to screening by ELISA.

6.5. Sequencing of Selected Phage

The amino acid sequence of phage inserts demonstrating a good ratio ofbinding to receptor domains and/or Caco-2 cells over background BSAbinding were deduced from the nucleotide sequence obtained by sequencing(Sequenase®, U.S. Biochemical Corp., Cleveland, Ohio) both DNA strandsof the appropriate region in the viral genome. The third round acideluate was screened by ELISA on HPT-1, BSA and Caco-2 fixed cells. Phagedesignated 5PAX were carried through five rounds of panning after whicha number of phages were sequenced prior to screening by ELISA.

One well of a 24 well plate was coated with 10 μg/ml of GIT receptor andthe plate was incubated overnight at 4° C. The plate was blocked with0.5 BSA-PBS for one hour. A mixture of the DC8, D38 and DC43 phagelibraries was added to the plate and the plate was incubated for 2 to 3hours at room temperature on a rotator. After washing the well 10 timeswith 1% BSA plus 0.05% Tween 20 in PBS, the well was eluted with 0.05mglycine, pH2. The phage was then eluted with 0.2M NaPO₄. The elutedphage was titered on agar plates; the remaining phage was amplifiedovernight. The next day the amplified phage was added to a second coatedplate and the panning procedure was repeated as described above. Theeluted phage from the second pan as well as the amplified phage from thefirst pan was titered on agar plates. Following amplification overnightof the phage from the second pan, the panning procedure was repeated asdescribed above. The phage eluted from the third pan and the amplifiedphage from the second pan were then titered overnight on agar plates.Isolated phage colonies were amplified overnight prior to use in anELISA assay.

6.6. Receptor ELISA Procedure

96 well plates were coated overnight with GIT receptor, BSA and,optionally, IDE (insulin degrading enzyme, an irrelevant His-fusionprotein)or hTF. The plates were blocked for one hour with 0.5% BSA-PBS.After clarification, the amplified phage were diluted 1:100 in 1% BSAplus 0.05% Tween 20 in PBS and added to the plates. Following incubationof the plates on a rotator for 1 to 2 hours, the plates were washed 5times with 1% BSA plus 0.05% Tween 20 in PBS. Dilute anti-M13-HRPconjugate (anti-M13 antibody linked to horse radish peroxidase (HRP))was added to all the wells and the plate was incubated for one hour on arotator. After the plates were washed 5 times, as described above, TMBsubstrate was added to the wells. The plates were read at 650 nmabsorbance.

Receptor ELISA Results:

Below are the results of ELISA assays which assessed the binding ofphage panned on the hSI receptor to microtiter plates coated with hSIand BSA. Table 1 shows the OD results as well as the ratio of hSI to BSAbinding.

TABLE 1 PHAGE hSI BSA hSI/BSA S15 0.478 0.053 9 S21 0.845 0.092 9 S220.399 0.061 7 SNi10 0.57 0.051 11 SNi28 0.942 0.113 8 SNi34 0.761 0.1157 SNi38 0.466 0.076 6 SNi45 0.518 0.056 9 SNiAX2 0.383 0.065 6 SNiAX60.369 0.056 7 SNiAX8 0.342 0.068 5 BLANK 0.063 0.042 2

Below are the results of an ELISA which assessed the binding of phagepanned on the D2H receptor to microtiter plates coated with D2H and BSA.Table 2 shows the OD results as well as the ratio of D2H to BSA binding.

TABLE 2 PHAGE D2H BSA D2H/BSA DAB3 0.406 0.072 6 DAB7 0.702 0.09 8 DAB100.644 0.153 4 DAB18 0.467 0.085 5 DAB24 1.801 0.441 4 DAB30 0.704 0.1216 DAX15 0.391 0.101 4 DAX23 0.698 0.153 5 DAX24 0.591 0.118 5 DAX271.577 0.424 4 BLANK 0.038 0.037 1

Below are the results of an ELISA which assessed the binding of phagepanned for two rounds on the D2H receptor followed by a third round panon Caco-2 snapwells. Binding to fixed Caco-2 cells, D2H and BSA wasexamined. Table 3 shows the OD results as well as the ratio of D2H toBSA binding.

TABLE 3 PHAGE Caco-2 D2H BSA D2H/BSA DCX8 0.498 0.163 0.063 3 DCX110.224 0.222 0.071 3 DCX26 0.114 0.956 0.213 4 DCX33 0.164 0.616 0.103 6DCX36 0.149 0.293 0.064 5 DCX39 0.121 0.299 0.066 5 DCX42 0.308 0.1580.065 2 DCX45 0.147 0.336 0.075 4 Blank 0.065 0.043 0.04 1

Below are the results of an ELISA which assessed the binding of phagepanned on the hPEPT1 receptor to hPEPT1 and BSA. Table 4 shows the ODresults as well as the ratio of hPEPT1 to BSA binding.

TABLE 4 PHAGE hPEPT1 BSA PEPT1/BSA PAX9 0.312 0.079 4 PAX14 1.102 0.1398 PAX15 0.301 0.079 4 PAX16 0.648 0.171 4 PAX17 0.514 0.095 5 PAX180.416 0.087 5 PAX35 0.474 0.065 7 PAX38 0.292 0.064 5 PAX40 0.461 0.0766 PAX43 0.345 0.069 5 PAX45 0.419 0.081 5 PAX46 0.429 0.077 6 P31 0.8070.075 11 P90 1.117 0.107 9 5PAX3 0.173 0.04 4 5PAX5 0.15 0.036 4 5PAX70.171 0.037 5 5PAX12 0.227 0.04 6 Blank 0.102 0.039 3

Table 5 shows the results of an ELISA which assessed the binding ofphage panned on the HPT-1 receptor to HPT-1 and BSA. The table shows theOD results as well as the ratio of HPT-1 to BSA binding.

TABLE 5 PHAGE HPT1 BSA HPT/BSA HAX9 0.382 0.075 5 HAX40 0.991 0.065 15HAX42 0.32 0.071 5

Table 6 shows the results of an ELISA which assessed the binding ofphage panned for two rounds on the HPT-1 receptor followed by a thirdround pan on Caco-2 snapwells. Binding to fixed Caco-2 cells, HPT-1 andBSA was examined. The table shows the OD results as well as the ratio ofHPT-1 to BSA binding.

TABLE 6 PHAGE Caco-2 HPT1 BSA HPT1/BSA HCA3 0.406 0.048 0.038 1Cell ELISA Procedure

Phage ELISA was used as described above with the following changes.Diluent and wash buffer was PBS containing 1% BSA and 0.05% Tween 20 andplates were washed five times at each wash step. Supernatant of infectedbacterial cultures was diluted 1:100 and incubated with protein coatedplates for 2–3 hours with mild agitation. Anti-M13 Horseradishperoxidase (HRP) conjugate (Pharmacia, Piscataway, N.J.) was diluted1:8000.

Fixed Caco-2, C2BBe1, and A431 cell plates were prepared by growingcells on tissue culture treated microtiter plates. When cells wereconfluent, plates were fixed with 10% formaldehyde, washed twice withPBS and stored with 0.5% BSA-PBS at −20° C. On the day of the assay,thawed plates were treated with PBS containing 0.1% phenylhydrazine forone hour at 37° C. followed by two PBS washes and blocking for One hourwith 0.5% BSA-PBS. The standard ELISA procedure was followed at thispoint.

Phage which showed specificity to a GIT receptor was furthercharacterized by ELISA on a variety of recombinant proteins. Phage whichcontinued to exhibit GIT receptor specificity was sequenced.

TABLE 7 TARGET BINDING PHAGE INSERT SEQUENCES: SEQ. ID. NO. hSI S15 1RSGAYESPDGRGGRSYVGGGGGCGNIGRKHNLWGLRTASPACWD S21 2SPRSFWPVVSRHESFGISNYLGCGYRTCISGTMTKSSPIYPRHS S22 3SSSSDWGGVPGKVVRERFKGRGCGISITSVLTGKPNPCPEPKAA SNi10 4RVGQCTDSDVRRPWARSCAHQGCGAGTRNSHGCITRPLRQASAH SNi28 5SHSGGHMRAYGDVFRELRDRWNATSHHTRPTPQLPRGPN SNi34 6SPCGGSWGRFMQGGLFGGRTDGCGAHRNRTSASLEPPSSDY SNi38 7RGAADQRRGWSENLGLPRVGWDAIAHNSYTFTSRRPRPP SNi45 8SGGEVSSWGRVNDLCARVSWTGCGTARSARTDNKGFLPKHSSLR SNiAX2 9SDSDGDHYGLRGGVRCSLRDRGCGLALSTVHAGPPSFYPKLSSP SNiAX4 10RSLGNYGVTGTVDVTVLPMPGHANHLGVSSASSSDPPRR SNiAX6 11RTTTAKGCLLGSFGVLSGCSFTPTSPPPHLGYPPHSVN SNiAX8 12SPKLSSVGVMTKVTELPTEGPNAISIPISATLGPRNPLR D2H DAB3 13RWCGAELCNSVTKKFRPGWRDHANPSTHHRTPPPSQSSP DAB7 14RWCGADDPCGASRWRGGNSLFGCGLRCSAAQSTPSGRIHSTSTS DAB10 15SKSGEGGDSSRGETGWARVRSHAMTAGRFRWYNQLPSDR DAB18 16RSSANNCEWKSDWMRRACIARYANSSGPARAVDTKAAP DAB24 17SKWSWSSRWGSPQDKVEKTRAGCGGSPSSTNCHPYTFAPPPQAG DAB30 18SGFWEFSRGLWDGENRKSVRSGCGFRGSSAQGPCPVTPATIDKH DAX15 19SESGRCRSVSRWMTTWQTQKGGCGSNVSRGSPLDPSHQTGHATT DAX23 20REWRFAGPPLDLWAGPSLPSFNASSHPRALRTYWSQRPR DAX24 21RMEDIKNSGWRDSCRWGDLRPGCGSRQWYPSNMRSSRDYPAGGH DAX27 22SHPWYRHWNHGDFSGSGQSRHTPPESPHPGRPNATI DCX8 23RYKHDIGCDAGVDKKSSSVRGGCGAHSSPPRAGRGPRGTMVSRL DCX11 24SQGSKQCMQYRTGRLTVGSEYGCGMNPARHATPAYPARLLPRYR DCX26 25SGRTTSEISGLWGWGDDRSGYGWGNTLRPNYIPYRQATNRHRYT DCX33 26RWNWTVLPATGGHYWTRSTDYHAINNHRPSIPHQHPTPI DCX36 27SWSSWNWSSKTTRLGDRATREGCGPSQSDGCPYNGRLTTVKPRT DCX39 28SGSLNAWQPRSWVGGAFRSHANNNLNPKPTMVTRHPT DCX42 29RYSGLSPRDNGPACSQEATLEGCGAQRLMSTRRKGRNSRPGWTL DCX45 30SVGNDKTSRPVSFYGRVSDLWNASLMPKRTPSSKRHDDG hPEPT1 PAX9 31RWPSVGYKGNGSDTIDVHSNDASTKRSLIYNHRRPLFP PAX14 32RTFENDGLGVGRSIQKKSDRWYASHNIRSHFASMSPAGK PAX15 33SYCRVKGGGEGGHTDSNLARSGCGKVARTSRLQHINPRATPPSR PAX16 34SWTRWGKHTHGGFVNKSPPGKNATSPYTDAQLPSDQGPP PAX17 35SQVDSFRNSFRWYEPSRALCHGCGKRDTSTTRIHNSPSDSYPTR PAX18 36SFLRFQSPRFEDYSRTISRLRNATNPSNVSDAHNNRALA PAX35 37RSITDGGINEVDLSSVSNVLENANSHRAYRKHRPTLKRP PAX38 38SSKVSSPRDPTVPRKGGNVDYGCGHRSSARMPTSALSSITKCYT PAX40 39RASTQGGRGVAPEFGASVLGRGCGSATYYTNSTSCKDAMGHNYS PAX43 40RWCEKHKFTAARCSAGAGFERDASRPPQPAHRDNTNRNA PAX45 41SFQVYPDHGLERHALDGTGPLYAMPGRWIRARPQNRDRQ PAX46 42SRCTDNEQCPDTGTRSRSVSNARYFSSRLLKTHAPHRP P31 43SARDSGPAEDGSRAVRLNGVENANTRKSSRSNPRGRRHP P90 44SSADAEKCAGSLLWWGRQNNSGCGSPTKKHLKHRNRSQTSSSSH 5PAX3 45RPKNVADAYSSQDGAAAEETSHASNAARKSPKHKPLRRP 5PAX5 46RGSTGTAGGERSGVLNLHTRDNASGSGFKPWYPSNRGHK 5PAX7 47RWGWERSPSDYDSDMDLGARRYATRTHRAPPRVLKAPLP 5PAX12 48RGWKCEGSQAAYGDKDIGRSRGCGSITKNNTNHAHPSHGAVAKI HPT-1 HAX9 49SREEANWDGYKREMSHRSRFWDATHLSRPRRPANSGDPN HAX35 50EWYSWKRSSKSTGLGDTATREGCGPSQSDGCPYNGRLTTVKPRK HAX40 51REFAERRLWGCDDLSWRLDAEGCGPTPSNRAVKHRKPRPRSPAL HAX42 52SDHALGTNLRSDNAKEPGDYNCCGNGNSTGRKVFNRRRPSAIPT HCA3 53RHISEYSFANSHLMGGESKRKGCGINGSFSPTCPRSPTPAFRRT H40 54SRESGMWGSWWRGHRLNSTGGNANMNASLPPDPPVSTP PAX2 55STPPSREAYSRPYSVDSDSDTNAKHSSHNRRLRTRSRPN

TABLE 8 DNA Sequences for Clones used in in vivo Pan S15 (SEQ ID NO: 56)TCTCACTCCTCGAGATCCGGCGCTTATGAGAGTCCGGATGGTCGGGGGGGTCGGAGCTATGTGGGGGGCGGGGGTGGNTGTGGTAACATTGGTCGGAAGCATAACCTGTGGGGGCTGCGTACCGCGTCGCCGGCCTGCTGGGACTCTAGAATCGAAGGTCGCGCTAGACCTTCGAGA S21 (SEQ ID NO:57) TCTCACTCCTCGAGTCCTCGCTCTTTCTGGCCCGTTGTGTCCCGGCATGAGTCGTTTGGGATCTCTAACTATTTGGGNTGTGGTTATCGTACATGTATCTCCGGCACGATGACTAAGTCTAGCCCGATTTACCCTCGGCATTCGTCTAGAATCGAAGGTCGCGCTAGACCTTCGAGA S22 (SEQ ID NO:58) TCTCACTCCTCGAGTAGTAGCTCCGATTGGGGTGGTGTGCCTGGGAAGGTGGTTAGGGAGCGCTTTAAGGGGCGCGGTTGTGGTATTTCCATCACCTCCGTGCTCACTGGGAAGCCCAATCCGTGTCCGGAGCCTAAGGCGGCCTCTAGAATCGAAGGTCGCGCTAGACCTTCGAGA SNi 10 (SEQ IDNO: 59) TCTCACTCCTCGAGAGTTGGCCAGTGCACGGATTCTGATGTGCGGCGTCCTTGGGCCAGGTCTTGCGCTCATCAGGGTTGTGGTGCGGGCACTCGCAACTCGCACGGCTGCATCACCCGTCCTCTCCGCCAGGCTAGCGCTCATTCTAGAATCGAAGGTCGCGCTAGACCTTCGAGA SNi 28 (SEQ IDNO: 60) TCTCACTCCTCGAGCCACTCCGGTGGTATGAATAGGGCCTACGGGGATGTGTTTAGGGAGCTTCGTGATCGGTGGAACGCCACTTCCCACCACACTCGCCCCACCCCTCAGCTCCCCCGTGGGCCTAATTCTAGAATCGAAGGTCGCGCTAGACCTTCGAGA SNi 34 (SEQ ID NO: 61)TCTCACTCCTCGAGTCCGTGCGGGGGGTCGTGGGGGCGTTTTATGCAGGGTGGCCTTTTCGGCGGTAGGACTGATGGTTGTGGTGCCCATAGAAACCGCACTTCTGCGTCGTTAGAGCCCCCGAGCAGCGACTACTCTAGAATCGAAGGTCGCGCTAGACCTTCGAGA SNi 38 (SEQ ID NO: 62)TCTCACTCCTCGAGGGGCGCCGCCGATCAGCGGCGGGGGTGGTCCGAGAACTTGGGGTTGCCTAGGGTGGGGTGGGACGCCATCGCTCACAATAGCTATACGTTCACCTCGCGCCGCCCGCGCCCCCCCTCTAGA SNi 45 (SEQ ID NO: 63)TCTCACTCCTCGAGCGGTGGGGAGGTCAGCTCCTGGGGCCGCGTGAATGACCTCTGCGCTAGGGTGAGTTGGACTGGTTGTGGTACTGCTCGTTCCGCGCGTACCGACAACAAAGGCTTTCTTCCTAAGCACTCGTCACTCCGCTCTAGAATCGAAGGTCGCGCTAGACCTTCGAGA SNi AX2 (SEQ IDNO: 64) TCTCACTCCTCGAGTGATAGTGACGGGGATCATTATGGGCTTCGGGGGGGGGTGCGTTGTTCGCTTCGTGATAGGGGTTGTGGTCTGGCCCTGTCCACCGTCCATGCTGGTCCCCCCTCTTTTTACCCCAAGCTCTCCAGCCCCTCTAGAATCGAAGGTCGCGCTAGACCTTCGAGA SNi AX4 (SEQ IDNO: 65) TCTCACTCCTCGAGGAGCTTGGGTAATTATGGCGTCACCGGGACTGTGGACGTGACGGTTTTGCCCATGCCTGGCCACGCCAACCACCTTGGTGTCTCCTCCGCCTCTAGCTCTGATCCTCCGCGGCGCTCTAGAATCGAAGGTCGCGCTAGACCTTCGAGA SNi AX6 (SEQ ID NO: 66)TCTCACTCCTCGAGAACTACGACGGCTAAGGGGTGTCTTCTCGGAAGCTTCGGCGTTCTTAGTGGGTGCTCATTTACGCCAACCTCTCCACCGCCCCACCTAGGATACCCCCCCCACTCCGTCAATTCTAGAATCGAAGGTCGCGCTAGACCTTCGAGA SNi AX8 (SEQ ID NO: 67)TCTCACTCCTCGAGCCCGAAGTTGTCCAGCGTGGGTGTTATGACTAAGGTCACGGAGCTGCCCACGGAGGGGCCTAACGCCATTAGTATTCCGATCTCCGCGACCCTCGGCCCGCGCAACCCGCTCCGCTCTAGAATCGAAGGTCGCGCTAGACCTTCGAGA DAB3 (SEQ ID NO: 68)TCTCACTCCTCGAGGTGGTGCGGCGCTGAGCTGTGCAACTCGGTGACTAAGAAGTTTCGCCCGGGCTGGCGGGATCACGCCAATCCCTCCACCCATCATCGTACTCCCCCGCCCAGCCAGTCCAGCCCTTCTAGAATCGAAGGTCGCGCTAGACCTTCGAGA DAB7 (SEQ ID NO: 69)TCTCACTCCTCGAGGTGGTGCGGCGCTGATGACCCGTGTGGTGCCAGTCGTTGGCGGGGGGGCAACAGCTTGTTTGGTTGTGGTCTTCGTTGTAGTGCGGCGCAGAGCACCCCGAGTGGCAGGATCCATTCCACTTCGACCAGCTCTAGAATCGAAGGTGCGCTAGACCTTCGAGA DAB10 (SEQ ID NO:70) TCTCACTCCTCGAGTAAGTCCGGGGAGGGGGGTGACAGTAGCAGGGGCGAGACGGGCTGGGCGAGGGTTCGGTCTCACGCCATGACTGCTGGCCGCTTTCGGTGGTACAACCAGTTGCCCTCTGATCGGTCTAGAATCGAAGGTCGCGCTAGACCTTCGAGA DAB18 (SEQ ID NO: 71)TCTCACTCCTCGAGGTCGAGCGCCAATAATTGCGAGTGGAAGTCTGATTGGATGCGCAGGGCCTGTATTGCTCGTTACGCCAACAGTTCGGGCCCCGCCCGCGCCGTCGACACTAAGGCCGCGCCCTCTAGAATCGAAGGTCGCGCTAGACCTTCGAGA DAB24 (SEQ ID NO: 72)TCTCACTCCTCGAGTAAGTGGTCGTGGAGTTCGAGGTGGGGCTCCCCGCAGGATAAGGTTGAGAAGACCAGGGCGGGTTGTGGTGGTAGTCCCAGCAGCACCAATTGTCACCCCTACACCTTTGCCCCCCCCCCGCAAGCCGGCTCTAGAATCGAAGGTCGCGCTAGACCTTCGAGA DAB30 (SEQ IDNO: 73) TCTCACTCCTCGAGTGGGTTCTGGGAGTTTAGCAGGGGGCTTTGGGATGGGGAGAAACCGTAAGAGTGTCCGGTCGGGTTGTGGTTTTCGTGGCTCCTCTGCTCAGGGCCCGTGTCCGGTCACGCCTGCCACCATTGACAAACACTCTAGAATCGAAGGTCGCGCTAGACCTTCGAGA DAX15 (SEQ IDNO: 74) TCTCACTCCTCGAGTGAGAGCGGGCGGTGCCGTAGCGTGAGCCGGTGGATGACGACGTGGCAGACGCAGAAGGGCGGTTGTGGTTCCAATGTTTCCCGCGGTTCGCCCCTCGACCCCTCTCACCAGACCGGGCATGCCACTACTTCTAGAATCGAAGGTCGCGCTAGACCTTCGAGA DAX23 (SEQ IDNO: 75) TCTCACTCCTCGAGGGAGTGGAGGTTTGCCGGGCCGCCGTTGGACCTGTGGGCGGGTCCGAGCTTGCCCTCTTTTAACGCCAGTTCCCACCCTCGCGCCCTGCGCACCTATTGGTCCCAGCGGCCCCGCTCTAGAATCGAAGGTCGCGCTAGACCTTCGAGA DAX24 (SEQ ID NO: 76)TCTCACTCCTCGAGGATGGAGGACATCAAGAACTCGGGGTGGAGGGACTCTTGTAGGTGGGGTGACCTGAGGCCTGGTTGTGGTAGCCGCCAGTGGTACCCCTCGAATATGCGTTCTAGCAGAGATTACCCCGCGGGGGGCCACTCTAGAATCGAAGGTCGCGCTAGACCTTCGAGA DAX27 (SEQ IDNO: 77) TCTCACTCCTCGAGTCATCCGTGGTACAGGCATTGGAACCATGGTGACTTCTCTGGTTCGGGCCAGTCACGCCACACCCCGCCGGAGAGCCCCCACCCCGGCCGCCCTAATGCCACCATTTCTAGAAT DCX8(SEQ ID NO: 78)TCTCACTCCTCGAGATATAAGCACGATATCGGTTGCGATGCTGGGGTTGACAAGAAGTCGTCGTCTGTGCGTGGTGGTTGTGGTGCTCATTNGTCGCCACCCCGCGCCGGCCGTGGTCCTCGCGGCACGATGGTTAGCAGGCTTTCTAGAATCGAAGGTCGCGCTAGACCTTCGAGA DCX11 (SEQ IDNO: 79) TCTCACTCCTCGAGTCAGGGCTCCAAGCAGTGTATGCAGTACCGCACCGGTCGTTTGACGGTGGGGTCTGAGTATGGTTGTGGTATGAACCCCGCCCGCCATGCCACGCCCGCTTATCCGGCGCGCCTGCTGCCACGCTATCGCTCTAGAATCGAAGGTCGCGCTAGACCTTCGAGA DCX26 (SEQ IDNO: 80) TCTCACTCCTCGAGTGGGCGGACTACTAGTGAGATTTCTGGGCTCTGGGGTTGGGGTGACGACCGGAGCGGTTATGGTTGGGGTAACACGCTCCGCCCCAACTACATCCCTTATAGGCAGGCGACGAACAGGCATCGTTATACGTCTAGAATCGAAGGTCGCGCTAGACCTTCGAGA DCX33 (SEQ IDNO: 81) TCTCACTCCTCGAGGTGGAATTGGACTGTCTTGCCCGCCACTGGCGGCCATTACTGGACGCGTTCGACGGACTATCACGCCATTAACAATCACAGGCCGAGCATCCCCCACCAGCATCCGACCCCTATCTCTAGAATCGAAGGTCGCGCTAGACCTTCGAGA DCX36 (SEQ ID NO: 82)TCTCACTCCTCGAGTTGGTCGTCGTGGAATTGGAGCTCTAAGACTACTCGTCTGGGCGACAGGGCGACTCGGGAGGGTTGTGGTCCCAGCCAGTCTGATGGCTGTCCTTATAACGGCCGCCTTACGACCGTCAAGCCTCGCACGTCTAGAATCGAAGGTCGCGCTAGACCTTCGAGA DCX39 (SEQ IDNO: 83) TCTCACTCCTCGAGTGGTAGTTTGAACGCATGGCAACCGCGGTCATGGGTGGGGGGCGCGTTCCGGTCACACGCCAACAATAACTTGAACCCCAAGCCCACCATGGTTACTNGTCACCCTACCTCTAGAATCGAAGGTCGCGCTAGACCTTCGAGA DCX42 (SEQ ID NO: 84)TCTCACTCCTCGAGGTATTCGGGTTTGTCCCCGCGGGACAACGGTCCCGCTTGTAGTCAGGAGGCTACCTTGGAGGGTTGTGGTGCGCAGAGGCTGATGTCCACCCGTCGCAAGGGCCGCAACTCCCGCCCCGGGTGGACGCTCTCTAGAATCGAAGGTCGCGCTAGACCCTTCGAGA DCX45 (SEQ IDNO: 85) TCTCACTCCTCGAGCGTGGGGAATGATAAGACTAGCAGGCCGGTTTCCTTCTACGGGCGCGTTAGTGATCTGTGGAACGCCAGCTTGATGCCGAAGCGTACTCCCAGCTCGAAGCGCCACGATGATGGCTCTAGAATCGAAGGTCGCGCTAGACCTTCGAGA PAAX2 (SEQ ID NO: 86)TCTCACTCCTCGAGTACTCCCCCCAGTAGGGAGGCGTATAGTAGGCCCTATAGTGTCGATAGCGATTCGGATACGAACGCCAAGCACAGCTCCCACAACCGCCGTNTGCGGACGCGCAGCCGCCCGAACTCTAGAATCGAAGGTCGCGCTAGACCTTCGAGA PAX9 (SEQ ID NO: 87)TCTCACTCCTCGAGATGGCCTAGTGTGGGTTACAAGGGTAATGGCAGTGACACTATTGATGTTCACAGCAATGACGCCAGTACTAAGAGGTCCCTCATCTATAACCACCGCCGCCCCNTCTTTCCCTCTAGAATCGAAGGTCGCGCTAGACCTTCGAGA PAX14 (SEQ ID NO: 88)TCTCACTCCTCGAGAACGTTTGAGAACGACGGGCTGGGCGTCGGCCGGTCTATTCAGAAGAAGTCGGATAGGTGGTACGCCAGCCACAACATTCGTAGCCATTTCGCGTCCATGTCTCCCGCTGGTAAGTCTAGAATCGAAGGTCGCGCTAGACCTTCGAGA PAX15 (SEQ ID NO: 89)TCTCACTCCTCGAGCTATTGTCGGGTTAAGGGTGGTGGGGAGGGGGGGCATACGGATTCCAATCTGGCTAGGTCGGGTTGTGGTAAGGTGGCCAGGACCAGCAGGCTTCAGCATATCAACCCGCGCGCTACCCCCCCCTCCCGGTCTAGAATCGAAGGTC PAX16 (SEQ ID NO: 90)TCTCACTCCTCGAGTTGGACTCGGTGGGGCAAGCACANTCATGGGGGGTTTGTGAACAAGTCTCCCCCTGGGAAGAACGCCACGAGCCCCTACACCGACGCCCAGCTGCCCAGTGATCAGGGTCCTCCCTCTAGAATCGAAGGTCGCGCTAGACCTTCGAGA PAX17 (SEQ ID NO: 91)TCTCACTCCTCGAGTCAGGTTGATTCGTTTCGTAATAGCTTTCGGTGGTATGAGCCGAGCAGGGCTCTGTGCCATGGTTGTGGTAAGCGCGACACCTCCACCACTCGTATCCACAATAGCCCCAGCGACTCCTATCCTACACGCTCTAGAATCGAAGGTCGCGCTAGACCTTCGAGA PAX18 (SEQ IDNO: 92) TCTCACTCCTCGAGCTTTTTGCGGTTCCAGAGTCCGAGGTTCGAGGATTACAGTAGGACGATCTNTCGGTTGCGCAACGCCACGAACCCGAGTAATGTCTCCGATGCGCACAATAACCGGGCCTTGGCCTCTAGAATCGAAGGTCGCGCTAGACCTTCGAGA PAX35 (SEQ ID NO: 93)TCTCACTCCTCGAGGAGCATCACCGACGGGGGCATCAATGAGGTGGACCTGAGTAGTGTGTCGAACGTTCTTGAGAACGCCAACTCGCATAGGGCCTACAGGAAGCATCGCCCGACCTTGAAGCGTCCTTCTAGAATCGAAGGTCGCGCTAGACCTTCGAGA PAX38 (SEQ ID NO: 94)TCTCACTCCTCGAGTTCGAAGGTGAGCAGCCCGAGGGATCCGACGGTCCCGCGGAAGGGCGGCAATGTTGATTATGGTTGTGGTCACAGGTCTTCCGCCCGGATGCCTACCTCCGCTCTGTCGTCGATCACGAAGTGCTACACTTCTAGAATCGAAGGTCGCGCTAGACCTTCGAGA PAX40 (SEQ IDNO: 95) TCTCACTCCTCGAGAGCCAGTANGCAGGGCGGCCGGGGTGTTGCCCCTGAGTTTGGGGCGAGCGTTTTGGGTNGTGGTTGTGGTAGCGCCACTTATTACACGAACTCCACCAGCTGCAAGGATGCTATGGGCCACAACTACTCGTCTAGAATCGAAGGTCGCGNTAGACCTTCGAGA PAX43 (SEQ IDNO: 96) TCTCACTCCTCGAGATGGTGCGAGAAGCACAAGTTTACGGCTGCGCGTTGCAGCGCGGGGGCGGGTTTTGAGAGGGANGCCAGCCGTCCGCCCCAGCCTGCCCACCGGGATAATACCAACCGTAATGCNTNTAGAATCGAAGGTCGCGCTAGACCTTCGAGA PAX45 (SEQ ID NO: 97)TCTCACTCCTCGAGTTTTCAGGTGTACCCGGACCATGGTCTGGAGAGGCATGCTTTGGACGGGACGGGTCCGCTTTACGCCATGCCCGGCCGCTGGATTAGGGCGCGTCCGCAGAACAGGGACCGCCAGTCTAGAATCGAAGGTCGCGCTAGACCTTCGAGA PAX46 (SEQ ID NO: 98)TCTCACTCCTCGAGCAGGTGTACGGACAACGAGCAGTGCCCCGATACCGGGANTAGGTCTCGTTCCGTTAGTAACGCCAGGTACTTTTCGAGCAGGTTGCTCAAGACTCACGCCCCCCATCGCCCTTCTAGAATCGAAGGTCGCGCTAGACCTTCGAGA P31 (SEQ ID NO: 99)TCTCACTCCTCGAGTGCCAGGGATAGCGGGCCTGCGGAGGATGGGTCCCGCGCCGTCCGGTTGAACGGGGTTGAGAACGCCAACACTAGGAAGTCCTCCCGCAGTAACCCGCGGGGTAGGCGCCATCCCTCTAGAATCGAAGGTCGCGCTAGACCTTCGAGA P90 (SEQ ID NO: 100)TCTCACTCCTCGAGTTCCGCCGATGCGGAGAAGTGTGCGGGCAGTCTGTTGTGGTGGGGTAGGCAGAACAACTCCGGTTGTGGTTCGCCCACGAAGAAGCATCTGAAGCACCGCAATCGCAGTCAGACCTCCTCTTCGTCCCACTCTAGAATCGAAGGTCGCGCTAGACCTTCGAGA 5PAX3 (SEQ IDNO: 101) TCTCACTCCTCGAGACCGAAGAACGTGGCCGATGCTTATTCGTCTCAGGACGGGGCGGCGGCCGAGGAGACGTCTCACGCCAGTAATGCCGCGCGGAAGTCCCCTAAGCACAAGCCCTTGAGGCGGCCTTCTAGAATCGAAGGTCGCGCTAGACCTTCGAGA 5PAX5 (SEQ ID NO: 102)TCTCACTCCTCGAGAGGCAGTACGGGGACGGCCGGCGGCGAGCGTTCCGGGGTGCTCAACCTGCACACCAGGGATAACGCCAGCGGCAGCGGTTTCAAACCGTGGTACCCTTCGAATCGGGGTCACAAGTCTAGAATCGAAGGTCGCGCTAGACCTTCGAGA 5PAX7 (SEQ ID NO: 103)TCTCACTCCTCGAGGTGGGGGTGGGAGAGGAGTCCGTCCGACTACGATTCTGATATGGACTTGGGGGCGAGGAGGTACGCCACCCGCACCCACCGCGCGCCCCCTCGCGTCTTGAAGGCTCCCCTGCCCTCTAGAATCGAAGGTCGCGCTAGACCTTCGAGA 5PAX12 (SEQ ID NO: 104)TCTCACTCCTCGAGGCACTGGAAGTGCGAGGGCTCTCAGGCTGCCTACGGGGACAAGGATATCGGGAGGTCCAGGGGTTGTGGTTCCATTACAAAGAATAACACTAATCACGCCCATCCTAGCCACGGCGCCGTTGCTAAGATCTCTAGAATCGAAGGTCGCGCTAGACCTTCGAGA HAX9 (SEQ ID No:105) TCTCACTCCTCGAGCCGCGAGGAGGCGAACTGGGACGGCTATAAGAGGGAGATGAGCCACCGGAGTCGCTTTTGGGACGCCACCCACCTGTCCCGCCCTCGCCGCCCCGCTAACTCTGGTGACCCTAACTCTAGAATCGAAGGTCGCGCTAGACCTTCGAGA HAX40 (SEQ ID NO: 106)TCTCACTCNTCGAGAGAGTTCGCGGAGAGGAGGTTGTGGGGGTGTGATGACCTGAGTTGGCGTCTCGACGCGGAGGGTTGTGGTCCCACTCCGAGCAATCGGGCCGTCAAGCATCGCAAGCCCCGCCCACGCTCCCCCGCACTCTCTAGAATCGAAGGTCGCGCTAGACCTTCGAGA HAX42 (SEQ IDNO: 107) TCTCACTCNTNGAGTGATCACGCGTTGGGGACGAATCTGAGGTCTGACAATGCCAAGGAGCCGGGTGATTACAACTGTTGTGGTAACGGGAACTCTACCGGGCGAAAGGTTTTTAACCGTAGGCGCCCCTCCGCCATCCCCANTTCTAGAATCGAAGGTCGCGCTAGACCTTCGAGA HCA3 (SEQ ID NO:108) TCTCACTCCTCGAGGCATATTTCTGAGTATAGCTTTGCGAATTCCCACTTGATGGGTGGCGAGTCCAAGCGGAAGGGTTGTGGTATTAACGGCTCCTTTTCTCCCACTTGTCCCCGCTCCCCCACCCCAGCCTTCCGCCGCACCTCTAGAATCGAAGGTCGCGCTAGACCTTCGAGA H40 (SEQ ID NO:109) TCTCACTCCTCGAGCCGGGAGAGCGGGATGTGGGGTAGTTGGTGGCGTGGTCACAGGTTGAATTCCACGGGGGGTAACGCCAACATGAATGCTAGTCTGCCCCCCGACCCCCCTGTTTCCACTCCGTCTAGAATCGAAGGTCGCGCTAGACCTTCGAG

By comparison of the amino acid sequences of the clones binding GITreceptors, certain sequence similarities or “motifs” were recognized.These motifs can often represent the part of the sequence that isimportant for binding to the target. Table 9 identifies regions ofsequence similarity or sequence motifs (in boldface) that wereidentified among GIT binding peptides (corresponding SEQ ID NOS. areshown in Table 7).

TABLE 9 PEPT-1 SEQ. ID. NO. HPT1 P31SARDSGPAEDGSRAVRLNGVENANTRKSSRSNPRGRRHP 43 PAX9RWPSVGYKGNGSDTIDVHSNDASTKRSLIYNHRRPLFP 31 HAX42SDHALGTNLRSDNAKEPGDYNCCGNGNSTGRK-VFNRRRPSAIPT 52 PAX2STPPSREAYSRPYSVDSDSDTNAKHSSHNRRLRTRSRPN 55 hSI SNi10RVGQCTDSDVRRPWARSCAHQGCGAGTRNSHGCITRPLRQASAH 4 SNi38RGAADQRRGWSENLGLPRVGWDAIAHNSYTFTSRRPRPP 7 S15RSGAYESPDGRGGRSYVGGGGGCGNIGRKHNLWGLRTASPACWD 1 SNi34SPCGGSWGRFMQGGLFGGRTDGCGAHRNRTSASLEPPSSDY 6 D2H DAB10SKSGEGGDSSRGETGWARVRSHAMTAGRFRWYNQLPSDR 15 DAB30SGFWEFSRGLWDGENRKSVRSGCGFRGSSAQGPCPVTPATIDKH 18 DCX8RYKHDIGCDAGVDKKSSSVRGGCG-AHSSPPRAGRGPRGTMVSRL 23Phage Binding to Caco-2 Cells

Phage expressing presumed GIT binding peptide inserts were also assayedby ELISA on fixed Caco-2 or C2BBe1 cells as follows. Cells were platedat 1×10⁵ cells/well on 100 μl culture media and incubated at 30° C. in5% CO₂ overnight. 100 μl 25% formaldehyde was added to each well for 15minutes. Contents of the wells were removed by inverting the plate. Theplate was then washed 3 times with DPBS. 0.1% phenylhydrazine DPBSsolution was added to each well and incubated for 1 hr at 37° C. Theplate was inverted and washed 3 times. The plate was blocked with 0.5%BSA-DPBS for 1 hr at room temperature. The plate was inverted and washed3 times with 1% BPT (PBS containing 1% BSA and 0.05% Tween20). Phagediluted with 1% BPT was added to wells containing fixed cells. Wellswithout phage added were used to determine background binding of the HRPconjugate. The plates were incubated 2–3 hours on a rotor at roomtemperature. Plates were washed as before. Plates were incubated withdilute anti-M13-HRP antibody in 1% BPT for 1 hour at room temperature.Following washing, TMB substrate was added and absorbance of the plateswere read at 650 nm. Table 10 shows the relative binding of phageencoding peptides to fixed Caco-2 cells.

TABLE 10 Relative binding of phage encoding peptides to fixed Caco-2cells Fixed Caco-2 Phage cell binding SNi10 ++ SNi34 + P31 ++ 5PAX5 ++PAX2 + HAX42 + DCX8 +++ DCX11 + H1 + M13mpl18 −In Vivo Phase Selection:

Further selection of phage expressing peptides capable of binding to theGIT or transporting the GIT was done as follows. The purified librarywas resuspended in a buffer, such as TBS or PBS, and introduced onto oneside of a tissue barrier, e.g., injected into the duodenum, jejunum,ileum, colon or other in vivo animal site using, for instance, a closedloop model or open loop model. Following injection, samples of bodilyfluids located across the tissue barrier, e.g., samples of the portalcirculation and/or systemic circulation, were withdrawn at predeterminedtime points, such as 0 to 90 minutes and/or 2 to 6 hours or more. Analiquot of the withdrawn sample (e.g., blood) was used to directlyinfect a host, e.g., E. coli, in order to confirm the presence of phage.The remaining sample was incubated, e.g., overnight incubation with E.coli at 37° C. with shaking. The amplified phage present in the culturecan be sequenced individually to determine the identity of peptidescoded by the phage or, if further enrichment is desired, can beprecipitated using PEG, and resuspended in PBS. The phage can then befurther precipitated using PEG or used directly for administration toanother animal using a closed or open GIT loop model system. Portal orsystemic blood samples are collected and the phage transported into suchcirculation systems is subsequently amplified. In this manner,administration of the phage display library with, if desired, repeatadministration of the amplified phage to the GIT of the animal,permitted the selection of phage which was transported from the GIT tothe portal and/or systemic circulation of the animal.

If desired, following administration of the phage display library to thetissue barrier (e.g., GIT) of the animal model, the corresponding regionof the tissue barrier can be recovered at the end of the proceduresgiven above. This recovered tissue can be washed repeatedly in suitablebuffers, e.g., PBS containing protease inhibitors and homogenized in,for example, PBS containing protease inhibitors. The homogenate can beused to infect a host, such as E. coli, thus permitting amplification ofphages which bind tightly to the tissue barrier (e.g., intestinaltissue). Alternatively, the recovered tissue can be homogenized insuitable PBS buffers, washed repeatedly and the phage present in thefinal tissue homogenate can be amplified in E. coli. This approachpermits amplification (and subsequent identification of the associatedpeptides) of phages which either bind tightly to the tissue barrier(e.g., intestinal tissue) or which are internalized by the cells of thetissue barrier (e.g., epithelial cells of the intestinal tissue). Thisselection approach of phage which bind to tissues or which areinternalized by tissues can be repeated.

Treatment of Animal Tissue Barriers In Vivo with Phase DisplayPopulations

The purified phage display library (random or preselected) was dilutedto 500 μl in PBS buffer and injected into the closed (or open)intestinal loop model (e.g., rat, rabbit or other species). At time 0and at successive time points after injection, a sample of either theportal circulation or systemic circulation was withdrawn. An aliquot ofthe withdrawn blood was incubated with E. coli, followed by plating forphage plaques or for transduction units or for colonies where the phagecodes for resistance to antibiotics such as tetracycline. The remainderof the withdrawn blood sample (up to 150 μl) was incubated with 250 μlof E. coli and 5 ml of LB medium or other suitable growth medium. The E.coli cultures were incubated overnight by incubation at 37° C. on ashaking platform. Blood samples taken at other time points (such as 15min, 30 min, 45 min, 60 min, up to 6 hours) were processed in a similarmanner, permitting amplification of phages present in the portal orsystemic circulation in E. coli at these times. Following amplification,the amplified phage was recovered by PEG precipitation and resuspendedin PBS buffer or TBS buffer. The titer of the amplified phage, beforeand after PEG precipitation, was determined. The amplified, PEGprecipitated phage was diluted to a known phage titer (generally between10⁸ and 10¹⁰ phage or plaque forming units (p.f.u.) per ml) and wasinjected into the GIT of the animal closed (or open) loop model. Bloodsamples were collected from portal and/or systemic circulation atvarious time points and the phage transported into the blood sampleswere amplified in E. coli as given above for the first cycle.Subsequently, the phage was PEG-precipitated, resuspended, titered,diluted and injected into the GIT of the animal closed (or open) loopmodel. This procedure of phage injection followed by collection ofportal and/or systemic blood samples and amplification of phagetransported into these blood samples can be repeated, for example, up to10 times, to permit the selection of phages which are preferentiallytransported from the GIT into the portal and/or systemic circulation.

6.7. Transport of Phage from Rat Lumen into the Portal and SystemicCirculation

Phage from random phage display libraries as well as control phage wereinjected into the lumen of the rat gastro-intestinal tract (in situ ratclosed loop model). Blood was collected over time from either thesystemic circulation or portal circulation and the number of phage whichwere transported to the circulation was determined by titering bloodsamples in E. coli.

The phage display libraries used in this study were D38 and DC43 inwhich gene III codes for random 38-mer and 43-mer peptides,respectively. As a negative control, the identical phage M13 mp18, inwhich gene III does not code for a “random” peptide sequence, was used.Both the library phages D38 and DC43 were prepared from E. coli, mixedtogether, dialyzed against PBS, precipitated using PEG/NaCl and wereresuspended in PBS buffer. The M13mp18 control was processed in asimilar manner. The titer of each phage sample was determined and thephage samples were diluted in PBS to approximately the same titers priorto injection into the rat closed loop model.

For sampling from the systemic circulation, approximately 15 cm of theduodenum of Wistar rats was tied off (closed loop model), approximately0.5 ml of phage solution was injected into the closed loop and blood(0.4 ml) was sampled from the tail vein at various times. The timepoints used (in min) were: 0, 15, 30, 45, 60, 90, 120, 180, 240 and 300minutes. For sampling from the portal circulation, the portal vein wascatheterized, approximately 15 cm of the duodenum was tied off (closedloop model), 0.5 ml of phage solution was injected into the closed loopand blood was sampled from the portal vein catheter at various times. Asthe portal sampling is delicate, sampling times were restricted to 15,30, 45 and 60 minutes, where possible. The volume of phage injected intoeach animal was as follows:

ANIMALS (15) VOLUME OF PHAGE INJECTED R1–R3 0.50 ml R4 0.43 ml R5–R150.45 mlThe estimated number of transported phage has been adjusted to accountfor differences in volume injected into each animal (using 0.5 ml as thestandard volume).

To investigate transport into the systemic circulation, animals R1, R2and R3 received the control phage M13mp18 and animals R4, R5, R6 and R7received the test phage D38/DC43 mix. To investigate transport into theportal circulation, animals R8, R9 and R10 received the control phageM13mp18 and animals R11, R12, R13 and R14 received the test phageD38/DC43 mix. Animal R15* received the combined phage samples fromanimals R4–R7 (see Table 11) which were sampled from the systemiccirculation on day one, followed by amplification in. E. coli, PEGprecipitation and resuspension in PBS. On subsequent analysis, the titerof this phage was found to be 100 times greater than the other phagesamples used for animals R8-R14. Thus, the data presented for animalR15* is adjusted down.

Approximately 0.4 ml of the blood was collected at each time point ineach model system. 30 μl of the collected blood (systemic) was mixedwith 100 μl of the prepared E. coli strain K91Kan, incubated at 37° C.for 30 min, and plated out for plaque formation using Top Agarose on LBplates. Various negative controls were included in the titeringexperiments. The following day, the number of plaque forming units wasdetermined. Similarly, 30 μl of the collected blood (portal) and serialdilutions (1:100, 1:1000) thereof was mixed with 100 μl of the preparedE. coli strain K91Kan, incubated at 37° C. for 30 min, and plated outfor plaque formation using Top Agarose on LB plates. The following day,the number of plaque forming units was determined.

In addition, approximately 300 μl of the collected blood from each timepoint (systemic and portal) was incubated with 5 ml of prepared E. colistrain K91Kan in modified growth media containing 5 mM MgCl₂/MgSO4 at37° C. overnight with shaking (to permit phage amplification). Thesamples were centrifuged and the cell pellet was discarded. Samples ofthe phage supernatant were collected, serially diluted (10⁻², 10⁻⁴,10⁻⁶, 10⁻⁸) in TBS buffer, and plated for plaques in order to determinethe number of plaque forming units present in the amplified phagesamples.

Furthermore, an aliquot of phage was removed from the “amplified”supernatants obtained from test animals R4–R7 (samples from each timepoint were used), combined, and precipitated using PEG for two hours.The precipitated phage was resuspended in PBS buffer and was injectedinto closed loop model of animal R15*, followed by portal sampling.

The number of phage transported from the closed loop model into thesystemic circulation is presented in Table 11 hereafter. The number ofphage transported from the closed loop model into the portal circulationis presented in Table 12 hereafter. These numbers are corrected forphage input difference and for volume input differences. Clearly, morephage are present in the portal samples than in the systemic samples,indicative of either hepatic or RES clearance and/or phage instabilityin the systemic circulation. In addition, the uptake of phage from theGIT into the portal circulation is quite rapid, with substantial numberof phages detected within 15 minutes. The results from the portalsampling experiments would also indicate that the kinetics of uptake ofphage from the D38/DC43 libraries is quicker than that of the controlphage. Thus, there may be preferential uptake of phage coding for randompeptide sequences from the GIT into the portal circulation. In the caseof animals R13, R14 and R15*, the % of the phage transported into thetitered blood sample within the limited time frame (30, 45 and 15 mins,respectively) was estimated as 0.13%, 1.1% and 0.013%, respectively.

TABLE 11 NUMBER OF PHAGE TRANSPORTED FROM THE CLOSED LOOP MODEL INTO THESYSTEMIC CIRCULATION Time (min) R1 R2 R3 R4 R5 R6 R7 0 0 0 0 0 0 0 0 150 1 9 0 0 1 7 30 2 1 0 0 46 1 11 45 10 4 2 1 32 0 20 60 63 19 21 1 114 021 90 104 20 18 3 115 0 22 120 94 24 27 0 64 0 6 180 94 12 23 1 413 0 0240 14 1 20 0 36 0 0 300 1 1 4 2 0 0 0 Total number of 382 83 124 8 8202 87 transported phage

Animals R1, R2 and R3 received the control phage M13mp18.

Animals R4, R5, R6 and R7 received the test phage D38/DC43 mix.

TABLE 12 NUMBER OF PHAGE TRANSPORTED FROM THE CLOSED LOOP MODEL INTO THEPORTAL CIRCULATION Time (min) R8 R9 R10 R11 R12 R13 R14 R15* 15 15 6 3 119 231,000 1,000,000 20,000 30 1 5 26 — 0 60,000 272,000 — 45 — 1 555 —1 — 1,240,000 — 60 — — — — 420,000 — — —

Animals R8, R9 and R10 received the control phage M13mp18.

Animals R11, R12, R13 and R14 received the test phage D38/DC43 mix.

Animal R15* received the combined phage samples from animals R4–R7 (seeTable 11) which were sampled from the systemic circulation on day one,followed by PEG precipitation and resuspension in PBS. On subsequentanalysis, the titer of this phage was found to be 100 times greater thanthe other phage samples used for animals R8–R14. Thus, the datameasuring phage transport into the portal circulation for animal R15* isadjusted down.

These studies demonstrated that both the control phage and the D38/DC43phages are transported over time from the lumen of the GIT into theportal and systemic circulation, as demonstrated by titering the phagetransported to the blood in E. coli. More phage were transported fromthe test phage samples into the portal circulation than thecorresponding control phage sample. In addition, the kinetics oftransport of the test phage into the portal circulation appeared toexceed that of the control phage. Phage from the D38/DC43 librarieswhich appeared in the systemic circulation of different animals (R4–R7)were pooled, amplified in E. coli, precipitated, and re-applied to thelumen of the GIT, followed by collection in the portal circulation andtitering in E. coli. These selected phage were also transported from thelumen of the GIT into the portal circulation. This in situ loop modelmay represent an attractive screening model in which to identify peptidesequences which facilitate transport of phage and particles from the GITinto the circulation.

Using this screening model system, a number of preselected phagelibraries now exist, including a one pass systemic phage library fromanimals R4–R7, a one-pass portal library from animals R11–R14, and a twopass, rapid transport, systemic-portal phage library SP-2 from animalR15*.

6.8. Transport of Phage from Preselected Phage Libraries from the RatLumen into the Portal and Systemic Circulation

Four preselected phage libraries, GI-D2H, GI-hSI, GI-HPT1 and GI-hPEPT1,were constructed by pooling phage previously selected by screeningrandom phage display libraries D38 and DC43 using the HPT1, HPEPT1, D2Hand hSI receptor or binding sites located in the GIT. The phage pools,preselected phage libraries are shown in Table 13. Note that thesequences for PAX2, HAX1, HAX5, HAX6, HAX10, H10 and HAX44 are the same.Also, the sequence for HAX40 is the same as that for H44. Thecorresponding SEQ ID NOS. are shown in Table 7.

TABLE 13 PRESELECTED PHAGE LIBRARIES D2H HSI HPT1 hPEPT1 DAB3 S15 HAX9PAX2 (H10) DAB7 S21 HAX35 PAX9 DAB10 S22 HAX40 (H44) PAX14 DAB18 SNi10HAX42 PAX15 DAB24 SNi28 HCA3 PAX16 DAB30 SNi34 HAX1 PAX17 DAX15 SNi38HAX5 PAX18 DAX23 SNi45 HAX6 PAX35 DAX24 SNiAX2 HAX10 PAX38 DAX27 SNiAX6H40 PAX40 DCX8 SNiAX8 M13mp18 PAX43 DCX11 M13mp18 PAX45 DCX26 PAX46DCX33 P31 DCX36 P90 DCX39 5PAX3 DCX42 5PAX5 DCX45 5PAX7 M13mp18 5PAX12H40 M13mp18Similar to methods described herein above, these preselected phagelibraries together with the negative control phage M13mp18 were injectedinto the rat closed loop model (6 animals per preselected phagelibrary), blood was collected over time from the portal circulation viathe portal vein and, at the termination of the experiment, a systemicblood sample was collected from the tail vein and the intestinal tissueregion from the closed loop was collected.

In particular, phages selected in vitro to each receptor or binding sitelocated in the GIT were amplified in E. coli, PEG-precipitated,resuspended in TBS and the titer of each phage sample was determined byplaquing in E. coli as described above. Subsequently, an equal number ofeach phage (8×10⁸ phage) for each receptor site was pooled into apreselected phage library together with the negative control phageM13mp18 and each preselected phage library was administered to 6 Wistarrats per library (rats 1–6; G1-D2H, rats 7–12; GI-hSI, rats 13–18;GI-hPEPT1, and rats 19–24; GI-HPT1). Using the in situ loop modeldescribed above, 0.5 ml of preselected phage library solution wasinjected into the tied-off portion of the duodenum/jejunum. Blood wascollected into heparinized tubes from the portal vein at 0, 15, 30, 45and 60 minutes. A blood sample was taken from the systemic circulationat the end of the experiment. Similarly, the portion of theduodenum/jejunum used for phage injection was taken at the end of theexperiment.

Thirty microliters of the collected portal blood (neat and 10⁻², 10⁻¹,10⁻⁶ dilutions) was added to 30 μl E. coli K91Kan cells (overnightculture) and incubated at 37° C. for 10 min. Subsequently, 3 ml of topagarose was added and the samples were plated for plaques. One hundredmicroliters of the collected portal blood was added to 100 μl of E. coliK91Kan. Five milliliters of LB medium was then added and the sampleswere incubated at 37° C. overnight in a rotating microbial incubator.The E. coli was removed by centrifugation and the amplified phagesupernatant samples were either titered directly or werePEG-precipitated, resuspended in TBS and titered. Following titration ofthe amplified phage, samples containing phage from each set of animalswere combined, adjusting the titer of each sample to the same titer, andwere plated for plaques on LB agar lates (22 cm² square plates). Either12,000 or 24,000 phage were lated for plaques.

Thirty microliters of the collected systemic blood (neat and 10⁻², 10⁻¹,10⁻⁶ dilutions) was added to E. coli K91Kan cells, incubated at 37° C.for 10 min. Three ml of top agarose was then added and the samples wereplated for plaques. One hundred microliters of the collected systemicblood was added to 100 μl of E. coli K91Kan, incubated at 37° C. for 10min. Five milliliters of LB medium was then added and the samples wereincubated at 37° C. overnight in a rotating microbial incubator. The E.coli was removed by centrifugation and the amplified phage supernatantsamples were either titered directly or were PEG-precipitated,resuspended in TBS and titered. Following titration of the amplifiedphage, samples containing phage from each set of animals were combined,adjusting the titer of each sample to the same titer, and were platedfor plaques on LB agar plates (22 cm² square plates). Either 12,000 or24,000 phage were plated for plaques.

The intestinal tissue portion used in each closed loop was excised. Thetissue was cut into small segments, followed by 3 washings in sterilePBS containing protease inhibitors, and homogenized in an Ultra thorexhomogeniser (Int-D samples). Alternatively, the tissue (in PBSsupplemented with protease inhibitors) was homogenized in an UltraThorex homogenizer, washed 3 times in PBS containing protease inhibitorsand resuspended in PBS containing protease inhibitors (Int-G samples).In each case, serial dilutions (neat and 10⁻², 10⁻⁴, 10⁻⁶ dilutions) ofthe tissue homogenate was titered in E. coli. In addition, an aliquot(100 μl) of the tissue homogenate was added to 100 μl of E. coli K91Kan,incubated at 37° C. for 10 min, followed by addition of 5 ml of LBmedium and incubation overnight at 37° C. in a rotating microbialincubator.

The phage amplified from the portal blood, systemic blood and intestinaltissue was plated for plaques. The plaques were transferred to Hybond-NNylon filters, followed by denaturation (1.5M NaCl, 0.5M NaOH),neutralization (0.5M TRIS-HCl, pH7.4, 1.5M NaCl), and washing in 2×SSCbuffer. The filters were air-dried, and the DNA was cross-linked to thefilter (UV crosslinking: 2 min, high setting). The filters wereincubated in pre-hybridization buffer (6×SSC, 5× Denhardt's solution,0.1% SDS, 20 g/ml yeast tRNA) at 40° C.–45° C. for at least 60 min.

Synthetic oligonucleotides, (22-mers), complimentary to regions codingfor the receptor or binding sites used to create the preselected phagelibrary, were synthesized (see Table 14 below).

TABLE 14 OLIGONUCLEOTIDES USED IN IN VIVO SCREEN SEQ. CLONE NAME OLIGOID. NO. S15 ^(5′)TCCGGACTCTCATAAGCGCCGG^(3′) 111 S21^(5′)ACAACGGGCCAGAAAGAGCGAG^(3′) 112 S22^(5′)ACACCACCCCAATCGGAGCTAC^(3′) 113 SNi10^(5′)TCAGAATCCGTGCACTGGCCAA^(3′) 114 SNi28^(5′)GCCCTATTCATACCACCGGAGT^(3′) 115 SNi34^(5′)CATCAGTCCTACCGCCGAAAAG^(3′) 116 SNi38^(5′)CGTATAGCTATTGTGAGCGATG^(3′) 117 SNi45^(5′)ACGCGCGGAACGAGCAGTACCA^(3′) 118 SNiAX2^(5′)CCATAATGATCCCCGTCACTAT^(3′) 119 SNiAX6^(5′)AGACACCCCTTAGCCGTCGTAG^(3′) 120 SNiAX8^(5′)AGCTCCGTGACCTTAGTCATAA^(3′) 121 DAB3 ^(5′)TGCACAGCTCAGCGCCGCACCA^(3′) 122 DAB7 ^(5′)ACGGGTCATCAGCGCCGCACCA ^(3′) 123 DAB10^(5′)TGTCACCCCCCTCCCCGGACTT ^(3′) 124 DAB18 ^(5′)ACTCGCAATTATTGGCGCTCGA^(3′) 125 DAB24 ^(5′)GTCTTCTCAACCTTATCCTGCG ^(3′) 126 DAB30^(5′)AAAGCCCCCTGCTAAACTCCCA ^(3′) 127 DAX15 ^(5′)CTGCGTCTGCCACGTCGTCATC^(3′) 128 DAX23 ^(5′)GTTAAAAGAGGGCAAGCTCGGA ^(3′) 129 DAX24^(5′)CCGAGTTCTTGATGTCCTCCAT ^(3′) 130 DAX27 ^(5′)TCCAATGCCTGTACCACGGATG^(3′) 131 DCX8 ^(5′)TCGCAACCGATATCGTGCTTAT^(3′) 132 DCX11^(5′)TGCATACACTGCTTGGAGCCCT^(3′) 133 DCX26^(5′)GAAATCTCACTAGTAGTCCGCC^(3′) 134 DCX33^(5′)GCGGGCAAGACAGTCCAATTCC^(3′) 135 DCX36^(5′)GAGCTCCAATTCCACGACGACC^(3′) 136 DCX39^(5′)GGTTGCCATGCGTTCAAACTAC^(3′) 137 DCX42^(5′)TCCCGCGGGGACAAACCCGAAT^(3′) 138 DCX45^(5′)CTGCTAGTCTTATCATTCCCCA^(3′) 139 PAX2^(5′)CTATCGACACTATAGGGCCTAC^(3′) 140 PAX9^(5′)TACCCTTGTAACCCACACTAGG^(3′) 141 PAX14^(5′)TTCTTCTGAATAGACCGGCCGA^(3′) 142 PAX15^(5′)CCACCACCCTTAACCCGACAAT^(3′) 143 PAX16^(5′)AGGGGGAGACTTGTTCACAAAC^(3′) 144 PAX17^(5′)CGGCTCATACCACCGAAAGCTA^(3′) 145 PAX18^(5′)ATCGTCCTACTGTAATCCTCGA^(3′) 146 PAX35^(5′)GACACACTACTCAGGTCCACCT^(3′) 147 PAX38^(5′)CCATAATCAACATTGCCGCCCT^(3′) 148 PAX40^(5′)CAAAACGCTCGCCCCAAACTCA^(3′) 149 PAX43^(5′)GTAAACTTGTGCTTCTCGCACC^(3′) 150 PAX45^(5′)CCATGGTCCGGGTACACCTGAA^(3′) 151 PAX46^(5′)GTTACTAACGGAACGAGACCTA^(3′) 152 P31^(5′)TGTTGGCGTTCTCAACCCCGTT^(3′) 153 P90^(5′)ACAACCGGAGTTGTTCTGCCTA^(3′) 154 5PAX3^(5′)TAAGCATCGGCCACGTTCTTCG^(3′) 155 5PAX5^(5′)TTATCCCTGGTGTGCAGGTTGA^(3′) 156 5PAX7^(5′)TATCAGAATCGTAGTCGGACGG^(3′) 157 5PAX12^(5′)CTTTGTAATGGAACCACAACCC^(3′) 158 HAX9^(5′)CGGTGGCTCATCTCCCTCTTAT^(3′) 159 HAX35^(5′)ATCAGACTGGCTGGGACCACAA^(3′) 160 HAX40^(5′)CACAACCTCCTCTCCGCGAACT^(3′) 161 HAX42^(5′)AGATTCGTCCCCAACGCGTGAT^(3′) 162 HCA3^(5′)GGGAATTCGCAAAGCTATACTC^(3′) 163 H40^(5′)CCCCGTGGAATTCAACCTGTGA^(3′) 164 M13 (positive)^(5′)GTCGTCTTTCCAGACGT^(3′) 165 M13 (negative)^(5′)CTTGCATGCCTGCAGGTCGAC^(3′) 166The oligonucleotides (5 pmol) were 5′end labelled with ³²P-ATP and T4polynucleotide kinase and approximately 2.5 pmol of labelledoligonucleotide was used in hybridization studies. Hybridizations wereperformed at 40–45° C. overnight in buffer containing 6×SSC, 5×Denhardt's solution, 0.1% SDS, 20 μg/ml yeast tRNA and the radiolabeledsynthetic oligonucleotide, followed by washings (20–30 min at 40–45° C.)in the following buffers: (i) 2×SSC/0.1% SDS, (ii) 1×SSC/0.1% SDS, (iii)0.1×SSC/0.1% SDS. The filters were air-dried and exposed forautoradiography for 15 hours, 24 hours or 72 hours.

Hybridization data indicated that all the oligonucleotide probes boundspecifically to their phage target except for the HAX9 probe whichapparently was not labeled. A negative control probe that hybridizedonly to M13mp18 DNA showed a weak to negative signal in all samplestested (data not shown).

Hybridization data for pools from each receptor group of rats wascompiled. Tables 15, 16, 17 and 18 show a representative compilation ofautoradiograph signals of the HS1, D2H, HPT1 and hPEPT1 receptor groups.These Tables show the phage absorption and uptake from the closed loopGIT model to portal and systemic circulation and phageabsorption/internalization to intestinal tissue. In these Tables, Int-Grefers to intestinal tissue homogenized prior to washing and recoverywhile Int-D refers to intestinal tissue washed prior to homogenizationand phage recovery. In all cases, leading phage candidates were presentin more than one animal.

TABLE 15 SUMMARY OF AUTORADIOGRAPH SIGNALS OF HSI ANIMAL STUDY PhagePortal Int.-G Int.-D S15 ++ +/− +/− S21 − − − S22 − −/+ − SNi-10 +++/+++ ++ SNi-28 − − − SNi-34 ++ − − SNi-38 ++ − − SNi-45 − − − SNiAX-2 − −− SNiAX-6 − − − SNiAX-8 − − − M13 ++++++ ++++++ ++++++ M13 nd* + − *notdetected

TABLE 16 SUMMARY OF AUTORADIOGRAPH SIGNALS OF D2H ANIMAL STUDY PhagePortal Int.-G Int.-D DAB3 +++ +/− −/+ DAB7 ++ ++ −/+ DAB10 ++++++ +/−−/+ DAB18 − − − DAB24 − − − DAB30 ++++ ++ +++ DAX15 − − − DAX23 −/+ +−/+ DAX24 − − − DAX27 − + − DCX8 +++++ +/− − DCX11 ++++++ ++ −/+ DCX26 −− − DCX33 +++ ++ ++ DCX36 − − − DCX39 − −/+ − DCX42 − − −/+ DCX45 − ++ −M13 (+) +++++ +++++ +++++ M13 (−) +/− −/+ −

TABLE 17 SUMMARY OF AUTORADIOGRAPH SIGNALS OF HPT1 ANIMAL STUDY PhageInt.-G Portal Systemic H40 − − ++++ HAX9 ND ND ND HAX35 − + − HAX40 − −− HAX42 − ++ ++ HCA3 − − − PAX2 − +++ ++++ M13 (+) ++++++ ++++++ ++++++M13 (−) − −−/+ −

TABLE 18 SUMMARY OF AUTORADIOGRAPH SIGNALS OF hPEPT1 ANIMAL STUDY PhageInt.-G Portal Systemic PAX2 − ++ − PAX9 ++ +++ − PAX14 − ++ − PAX15 −/+− − PAX16 − − − PAX17 + ++/+ − PAX18 − − − PAX35 − − − PAX38 −/+ − −PAX40 + +++ − PAX43 + − − PAX45 − − − PAX46 − +++ − P31 ++ ++++ ++ 5PAX3++/+ ++ − 5PAX5 − − ++ 5PAX7 +++ − − 5PAX12 ++++ ++ − H40 ++ ++ − M13(+) ++++++ ++++++ ++++++ M13 (−) − − −Apart from the synthetic oligonucleotide to HAX9, all oligonucleotideswere initially confirmed to be radiolabeled, as determined byhybridization to the corresponding phage target (eg., phage S15hybridized to the oligonucleotide S15). In addition, under theexperimental conditions used, the oligonucleotides essentially did nothybridize to the negative control phage template M13mp18. Twooligonucleotides were synthesized to the phage M13mp18: (1) a positiveoligonucleotide which hybridizes to a conserved sequence in both M13mp18and each of the GIT receptor or GIT binding site selected phages[designated M13 (positive)]; and (2) a negative oligonucleotide whichonly hybridizes to a sequence unique to the multiple cloning site ofphage M13mp18 and which does not hybridize to any of the GIT receptor orGIT binding site selected phages.

In the case of the hSI pool of phages, only four phages were transportedfrom the closed loop model into the portal circulation: phages S15,SNi-10, SNi-34 and SNi-38. The other phages, S21, S22, SNi-28, SNi-45,SNiAX-2, SNiAX-6 and SNiAX-8, were not transported from the GIT into theportal circulation. In addition, phages SNi-10 and to a lesser extentphages S15 and S22 were found in the intestine samples or fractions,whereas the other phages were not. There was a very low presence (<0.1%)of the phage M13mp18 in the Int-G samples. These results show thatphages can be further selected from pre-selected libraries, permittingthe identification of phages which are transported from the GIT closedloop into the portal circulation or phages which bind to or areinternalized by intestinal tissue.

In the case of the D2H pool of phages, there was a rank order by whichphages were transported from the GIT closed loop model into the portalcirculation, with phages DCX11 and DAB10 preferably transported,followed by phages DCX8, DAB30, DAB3 and DAB7. A number of phages fromthis pool were not transported into the portal circulation, includingphages DAB18, DAB24, DAX15, DAX24, DAX27, DCX26, DCX36, DCX39, DCX42,DCX45. There is a very low level of transport of phage DAX23 from theGIT into the portal circulation. Similarly, only some of the phages werefound in the intestinal samples fractions, including phages DAB30,DCX33, DAB7, DCX11, DCX45 and to a much lesser extent phages DAB3,DAB10, DCX8, DCX39, DCX42. Some phages were not found in the intestinalsamples, including phages DAB18, DAB24, DAX15, DAX24, DCX26, and DCX36.There was a very low presence (<0.1%) of the phage M13mp18 in the Int-Gsamples. These results showed that phages can be further selected frompre-selected libraries, permitting the identification of phages whichare transported from the GIT closed loop into the portal circulation orphages which bind to or are internalized by intestinal tissue.

In the case of the HPT1 pool of phages, there was a rank order by whichphages were transported from the GIT closed loop model into the portalor systemic circulation. Phage PAX2 (which was used at a 4×concentration relative to the other phages in this pool) followed byphage HAX42 was found in the portal and systemic circulation; phage H40was found in the systemic circulation only. None of the phages in thispool were found in the intestine samples or fractions. Phage M13mp18 wasnot found in the intestine fractions or systemic circulation, with verylow incidence (<0.001%) in the portal circulation. These results showthat phages can be further selected from pre-selected libraries,permitting the identification of phages which are transported from theGIT closed loop into the portal and/or systemic circulation or phageswhich bind to or are internalized by intestinal tissue.

In the case of the hPEPT1 pool of phages, the phages PAX2 and H40 werealso included in this pool. A number of phages from this pool were foundin the portal circulation, including phages P31 (SEQ ID NO:43), PAX46,PAX9, H40, PAX17, PAX40, PAX2, PAX14, 5PAX3 and 5PAX12. A number ofphages were not found in the portal blood including the negative controlphage M13mp18, PAX15, PAX16, PAX18, PAX35, PAX38, PAX43, PAX45, P90,5PAX5 and 5PAX7. The only phage found in the systemic circulation werephages 5PAX5 and P31 (SEQ ID NO:43). In addition, there was preferentialbinding of some phages to the intestine, including phages 5PAX12, 5PAX7,5PAX3, H40, P31 (SEQ ID NO:43), PAX9, and to a lesser extent phagesPAX38 and PAX15. Some phages were not found in the intestine samples,including the negative control phage M13mp18 and the phages PAX2, PAX14,PAX16, PAX18, PAX35, PAX45, PAX46, P90 and 5PAX5. These results showthat phages can be further selected from pre-selected libraries,permitting the identification of phages which are transported from theGIT closed loop into the portal and/or systemic circulation or phageswhich bind to or are internalized by intestinal tissue.

Further Characterization of Select Sequences

Following initial screening of the four recombinant receptor sites(hPEPT1, HPT1, D2H, hSI) of the gastrointestinal tissue, with the phagedisplay libraries, a series of phage were isolated which showedpreferential binding to the respective target receptor sites incomparison to negative control protein BSA protein and the recombinantprotein recombinant human tissue factor (hTF) (which, like therecombinant receptors of the gastrointestinal tissue, contained apoly-histidine tag at its NH₂-terminal end). In subsequent experimentssame titers of the selected phage which bound to each target receptorsite were combined into a single pool (i.e., one pool of HPT1 bindingphage, one pool of hPEPT1 binding phage, one pool of D2H binding phage,and one pool of hSI binding phage). Each pool was supplemented with anequivalent titer of the negative control phage M13mp18. These phagepools were injected into a closed duodenal loop region of rat intestinaltissue and subsequently phage was harvested and recovered which wasbound to and retained by the intestinal tissue and/or was absorbed fromthe intestinal loop into the portal and/or systemic circulation. Inaddition, a selection of the initial phages which bound to the targetrecombinant receptor site were analyzed for binding to either fixedCaco-2 cells and/or to fixed C2BBe1 cells. The selection of the finallead peptide sequences was based on the ability of the phage, coding forthat peptide sequence (1) to bind to the target recombinant receptorsite in vitro in preference to its binding to the negative controlproteins BSA and/or hTFs, (2) to bind to rat intestinal tissue followinginjection into a closed duodenal loop of rat intestinal tissue inpreference to the negative control phage M13mp18, (3) to be absorbedfrom rat intestinal tissue into either the portal and/or systemiccirculation following injection into a closed duodenal loop of ratintestinal tissue in preference to the negative control phage M13mp18,and (4) to bind to either fixed Caco-2 cells or fixed C2BBe1 cells inphage binding studies in preference to the negative control phageM13mp18. Peptides were also selected with consideration to the ease ofchemical synthesis.

6.9. GST Fusion Proteins of GIT Targeting Peptides Construction of GSTFusion Proteins of GI Targeting Peptides

Glutathione S-transferase (GST) vectors encoding fusion proteins of GItargeting peptides were constructed in the vector pGEX4T-2 (source,Pharmacia Biotech, Piscataway, N.J.). Briefly, single-strand DNA fromthe clones of interest were amplified by the polymerase chain reaction.The amplified DNA was then cleaved with the restriction enzymes XhoI andNotI and then ligated into SalI/NotI cleaved pGEX4T-2. Followingtransformation, the DNA sequence for each construct was verified bysequencing.

For construction of the truncated versions of the GST fusion proteins,where the inserted sequence was less than 45 base pairs, overlappingoligonucleotides containing cohesive SaI and NotI termini, and encodingthe sequence of interest, were annealed and then ligated directly intoSalI/NotI cleaved pGEX4T-2. Following transformation, the DNA sequencefor each construct was verified.

A diagrammatic representation of the various GST fusion proteinconstructs that have been synthesized is indicated in FIGS. 5A–5C.

Expression and Purification of GST Fusion Proteins

Escherichia coli BL21 cells containing GST fusion protein constructswere grown overnight in 2×YT media containing 100 μg/ml ampicillin(2×YT/amp). Overnight cultures were diluted 1:100 in 2×YT broth (100ml), and cells were grown to an A₆₀₀ of 0.5 at 30° C., induced with 1 mMisopropyl-1-thio-B-D-galactopyranoside, and grown for an additional 3 h.Cells were harvested by centrifugation and resuspended in 5 ml of PBScontaining a mixture of the proteinase inhibitors (Boehringer/Mannheim).Cells were sonicated on ice, and the cell lysates were centrifuged at12,000×g for 10 minutes at 4° C. Supernatant fractions were reacted for30 minutes at room temperature with 2 ml of a 50% slurry ofglutathione-Sepharose® 4B, washed 3 times with 1.5 ml of PBS (at roomtemperature), and the bound GST fusion proteins were eluted by reactionfor 10 minutes at room temperature with 3×1 ml of 10 mM reducedglutathionein 50 mM Tris HCl pH 8.0. Protein was quantified by theBio-Rad protein assay followed by characterization by SDS-polyacrylamide gel electrophoresis.

ELISA of GST Fusion Peptides

The standard ELISA procedure was modified as follows. GST proteins werediluted to an appropriate concentration in PBS containing 1% BSA and0.05% Tween20 (1% BPT), titered and incubated one hour at roomtemperature. Following five washes an anti-GST monoclonal antibody wasadded (Sigma, St. Louis Clone GST-2 diluted 1:10,000 in 1% BPT) andincubated one hour. After five more washes goat anti-mouse IgG2b-HRP wasadded (Southern Biotechnology Associates Inc., Birmingham, Ala., diluted1:4000 in 1% BPT) and incubated one hour. After five washes plates weredeveloped with TMB peroxidase substrate (Kirkegard and Perry,Gaithersburg, Md.). All data is presented with background bindingsubtracted.

FIG. 6 shows the binding of GST-SNi10, GST-SNi34 and GST alone to thehSI receptor and to fixed C2BBe1 cells.

GST Fusion Proteins of Selected GIT Targeting Peptides

Results show that GST-DXB8, GST-PAX2, GST-P31, GST-SNi10 and GST-SNi34bound fixed Caco-2 or C2BBe1 cells (FIGS. 7 and 8) relative to GSTcontrol binding. GST-HAX42, GST-5PAX5, all showed weak to moderatebinding relative to GST control.

Interestingly, P31 truncation 103-GST (SEQ ID NO: 185) fusion proteinbound almost as well as full-length P31 (SEQ ID NO:43) to fixed Caco-2cells (A). This suggests the portion of the P31 sequence (SEQ ID NO:43)responsible for binding resides in this portion. PAX2.107 boundsimilarly to full-length PAX2; therefore, this portion most likelycontains the amino acid sequence responsible for binding (B). Inpreliminary assays, none of the DCX8 truncations bound similarly tofull-length DCX8 to Caco-2 cells suggesting the binding region spansmore than one of these pieces.

Inhibition of Binding by Synthetic Peptides Binding of GST-P31 to FixedC2BBe1 Cells

The standard ELISA procedure was modified as follows. GST fusionproteins and peptides were diluted to an appropriate concentration inPBS containing 1% BSA and 0.05% Tween 20. Peptides were titered, aconstant concentration of diluted GST protein was added to titeredpeptides and the mixture was incubated one hour at room temperature.Following five washes, an anti-GST monoclonal antibody was added (Sigma,St. Louis Clone GST-2 diluted 1:10,000 in 1% BPT) and incubated onehour. After five more washes goat anti-mouse IgG2b-HRP was added(Southern Biotechnology Associates Inc., Birmingham, Ala., diluted1:4000 in 1% BPT) and incubated one hour. After five washes plates weredeveloped with TMB peroxidase substrate (Kirkegard and Perry,Gaithersburg, Md.). All data is presented with background bindingsubtracted.

FIGS. 9A and 9B show the inhibition of GST-P31 binding to C2BBe1 fixedcells. The peptide competitors are ZElan024 (SEQ ID NO:288) which is thedansylated peptide version of P31 (SEQ ID NO:43) and ZElan044 (SEQ IDNO:310), ZElan049 (SEQ ID NO:315) and ZElan050 (SEQ ID NO:316) which aretruncated, dansylated pieces of P31 (SEQ ID NO:43). Data is presented asO.D. vs. peptide concentration and as percent inhibition of GST-P31binding vs. peptide concentration. Uncompeted GST-P31 binding wasconsidered as 100% binding. IC₅₀ values are estimates using the 50% lineon the percent inhibition graph.

GST-P31 and GST-PAX2 exhibited no crossreactive binding to ZElan024(P31) (SEQ ID NO:286) and ZElan018 (PAX2) (SEQ ID NO:281) at the 0.5μg/ml concentration used in competition assays. GST-HAX42 exhibitedcrossreactivity to ZElan018 (PAX2) (SEQ ID NO:281) and ZElan021 (HAX42)(SEQ ID NO:281) at the 5 μg/ml concentration used in competition assays.

FIGS. 10A–10C present a compilation of data generated by competitionELISA of GST-P31, GST-PAX2, GST-SNi10 and GST-HAX42 versus variousdansylated peptides on fixed C2BBe1 cells. IC₅₀ values are in μM andinclude ranges determined from multiple assays. The GST/C2BBe1 column isa summary of GST protein binding to fixed C2BBe1 cells.

Binding to Fixed Caco-2 Cells

Caco-2 cells were fixed, treated with phenylhydrazin and blocked asdescribed above. Synthetic peptides (100 μg/ml) were applied induplicate to Caco-2 cells and serially diluted down the 96-well plate.The corresponding GST-peptide fusion protein (10 μg) was added to eachwell and the plates were incubated for 2h at room temperature withagitation. Binding of the GST-peptide fusion proteins to the cells wasassayed using the ELISA technique described above. GST-P31 binding wasinhibited by ZElan024 (SEQ ID NO:288), ZElan028 (SEQ ID NO:294) andZElan031 (SEQ ID NO:297) as well as the two D forms ZElan053 (SEQ IDNO:319) and ZElan054 (SEQ ID NO:320). GST-PAX2 binding was inhibited byZElan032 (SEQ ID NO:298), ZElan033 (SEQ ID NO:299), and ZElan035 (SEQ IDNO:301). GST-HAX42 binding was not inhibited by ZElan021 (SEQ ID NO:285)(full length HAX42) but it was inhibited by ZElan018 (SEQ ID NO:281)(PAX2) and ZElan026 (SEQ ID NO:290) and ZElan038 (SEQ ID NO:304)(scrambled PAX2 peptides).

Transport and Uptake of GST-Peptide Fusions into Live Caco-2 Cells

Transport and uptake of GST-peptide fusions and deletion derivativesacross cultured polarized Caco-2 monolayers over 4 hours in HBSS bufferwas examined using an anti-GST ELISA assay. In another experiment,transport and uptake of GST-peptide fusions and deletion derivativesacross cultured polarized Caco-2 monolayers over 24 hours in serum-freemedium (SFM) was examined using an anti-GST ELISA assay.

Materials

Buffered Hank's balanced salt solution (bHBSS)=1×HBSS (GibcoCN.14065-031) supplemented with 0.011M glucose (1 g/l), 25 mM Hepes (15mM acid (3.575 g/l; Sigma CN.H3375); 10 mM base (2.603 g/l; SigmaCN.H1016)].

Chloroquine: Made up as 10 mM solution in water [Sigma CN C6628]

Lysate buffer: 30 mM Tris-HCl pH8.0; 1 mM EDTA

Serum-free medium (SFM) is normal medium without serum.

Method

a) 4h HBSS study: Transepithelial electrical flux (TER) across theCaco-2 monolayers grown on snapwells (passage 33; 23 days old) wasmeasured to confirm monolayer integrity before beginning the experiment.The medium was removed and the cells were washed once with bHBSS. bHBSScontaining 100 μM chloroquine was added and the cells were incubated for2h at 37° C. The bHBSS+chloroquine was replaced with 0.5 ml bHBSScontaining GST-peptide fusions (100 μg/ml) and the cells were incubatedas before. Basolateral samples were removed at the following times: 0,0.5h, 2h, and 4h. At 4h, TER was measured, the apical medium was sampledand the apical reservoir was washed 6 times with HBSS. The cells wereallowed to lyse for 1 h on ice in lysate buffer, after which, lysatesample was collected. All samples were stored at −70° C. until assay byanti-GST ELISA. Before analysis, samples were normalized for proteincontent relative to each other using a BioRad protein assay.

b) 24h SFM study: Transepithelial electrical flux (TER) across theCaco-2 monolayers grown on snapwells (passage 33; 23 days old) wasmeasured to confirm monolayer integrity before beginning the experiment.The medium was removed and the cells were washed once with SFM. SFMcontaining GST-peptide fusions (100 μg/ml) was added to the cells whichwere incubated at 37° C. for 24h at 5% CO2. After 24 hours, TER readingswere taken, and samples from the basolateral and apical reservoirs wereremoved. The apical reservoir was washed 6 times with PBS. The cellswere allowed to lyse for 1 h on ice in lysate buffer, after which lysatesample was collected. All samples were stored at −70° until assay byanti-GST ELISA. Before analysis, samples were normalized for proteincontent relative to each other using a BioRad protein assay.

Results

All of the GST-peptide fusions and controls examined were transportedacross live Caco-2 monolayers. Full-length GST-P31 and GST-DCX8, but nottruncations of these molecules had a higher flux than GST alone.

Internalization of GST-peptide fusions into polarized Caco-2 cells wasinvestigated in two experiments. In experiment 1, 15 μg of GST-peptidefusion was applied in bHBSS and internalized GST-peptide was recoveredby lysing the cells after 4h. In experiment 2, 10 μg of GST-peptide wasapplied in either a) bHBSS (lysate recovered after 4h), or b) serum-freemedium (lysate recovered after 24h).

FIG. 11A describes complete transport of GST-peptide across a polarizedCaco-2 monolayer and does not necessarily refer to internalization,i.e., the GST-peptide was recovered from the basolateral reservoir of asnapwell but the proteins could have crossed the barrier by theparacellular route.

Effect of Thrombin Cleavage on Binding of GST-Peptide Fusions to FixedCaco-2 Cells

Binding of intact and thrombin-cleaved GST-peptide fusions to fixedCaco-2 cells was compared. Reduced binding of the thrombin-cleavedGST-peptide fusions relative to intact fusions indicates that thepeptide component of the fusion, and not the GST domain, mediatesbinding.

Method

Confluent Caco-2 monolayers grown in 96-well plates (p38) were fixed andtreated with 0.1% phenylhydrazine before blocking with 0.1% BSA in PBS.Thirty micrograms of each GST-peptide was treated with bovine thrombin(1 μ/ml; 0.4 NIH units; Sigma CN.T9681) for 18h at room temperature in20 mM Tris-HCl pH8.0, 150 mM NaCl, 2.5 mM CaCl₂. Controls were similarlytreated without addition of thrombin. Ten micrograms of each GST-peptidefusion was removed for PAGE analysis, and 10 μg of fusions were added induplicate to the fixed Caco-2 cells before 5-fold serial dilutions (1%BPT diluent). The fusions were allowed to bind for 1h at roomtemperature. Following 6 washes with 1% BPT, binding was assayed byELISA.

Results

Results are shown in FIG. 12.

Conclusions:

PAGE analysis confirmed that the GST-peptide fusions were effectivelycleaved with thrombin. Cleavage with thrombin significantly reduceddetection of binding of GST-P31.103 (SEQ ID NO: 183), GST-PAX2.106 (SEQID NO: 188), GST-DCX8, GST-SNi10 to fixed Caco-2 cells, indicating thatthe peptide component, and not the GST domain, mediates binding.

6.10. Synthesis of Peptides

6.10.1. Procedure for Solid Phase Synthesis

Peptides may be prepared by methods that are known in the art. Forexample, in brief, solid phase peptide synthesis consists of couplingthe carboxyl group of the C-terminal amino acid to a resin andsuccessively adding N-alpha protected amino acids. The protecting groupsmay be any known in the art. Before each new amino acid is added to thegrowing chain, the protecting group of the previous amino acid added tothe chain is removed. The coupling of amino acids to appropriate resinsis described by Rivier et al., U.S. Pat. No. 4,244,946. Such solid phasesyntheses have been described, for example, by Merrifield, 1964, J. Am.Chem. Soc. 85:2149; Vale et al., 1981, Science 213:1394–1397; Marki etal., 1981, J. Am. Chem. Soc. 103:3178 and in U.S. Pat. Nos. 4,305,872and 4,316,891. In a preferred aspect, an automated peptide synthesizeris employed.

By way of example but not limitation, peptides can be synthesized on anApplied Biosystems Inc. (“ABI”) model 431A automated peptide synthesizerusing the “Fastmoc” synthesis protocol supplied by ABI, which uses2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(“HBTU”) (R. Knorr et al., 1989, Tet. Lett., 30:1927) as coupling agent.Syntheses can be carried out on 0.25 mmol of commercially available4-(2′,4′-dimethoxyphenyl-(9-fluorenyl-methoxycarbonyl)-aminomethyl)-phenoxypolystyrene resin (“Rink resin” from Advanced ChemTech) (H. Rink, 1987,Tet. Lett. 28:3787). Fmoc amino acids (1 mmol) are coupled according tothe Fastmoc protocol. The following side chain protected Fmoc amino acidderivatives are used: FmocArg(Pmc)OH; FmocAsn(Mbh)OH; FmocAsp(^(t)Bu)OH;FmocCys(Acm)OH; FmocGlu(^(t)Bu)OH; FmocGln(Mbh)OH; FmocHis(Tr)OH;FmocLys(Boc)OH; FmocSer(^(t)Bu)OH; FmocThr(^(t)Bu)OH; FmocTyr(^(t)Bu)OH.[Abbreviations: Acm, acetamidomethyl; Boc, tert-butoxycarbonyl; ^(t)Bu,tert-butyl; Fmoc, 9-fluorenylmethoxycarbonyl; Mbh,4,4′-dimethoxybenzhydryl; Pmc, 2,2,5,7,8-pentamethylchroman-6-sulfonyl;Tr, trityl].

Synthesis is carried out using N-methylpyrrolidone (NMP) as solvent,with HBTU dissolved in N,N-dimethylformamide (DMF). Deprotection of theFmoc group is effected using approximately 20% piperidine in NMP. At theend of each synthesis the amount of peptide present is assayed byultraviolet spectroscopy. A sample of dry peptide resin (about 3–10 mg)is weighed, then 20% piperidine in DMA (10 ml) is added. After 30 minsonication, the UV (ultraviolet) absorbance of thedibenzofulvene-piperidine adduct (formed by cleavage of the N-terminalFmoc group) is recorded at 301 nm. Peptide substitution (in mmol g⁻¹)can be calculated according to the equation:substitution=

$\frac{A \times v}{7800 \times w} \times 1000$where A is the absorbance at 301 nm, v is the volume of 20% piperidinein DMA (in ml), 7800 is the extinction coefficient (in mol⁻¹dm³ cm⁻¹) ofthe dibenzofulvene-piperidine adduct, and w is the weight of thepeptide-resin sample (in mg).

Finally, the N-terminal Fmoc group is cleaved using 20% piperidine inDMA, then acetylated using acetic anhydride and pyridine in DMA. Thepeptide resin is thoroughly washed with DMA, CH₂Cl₂ and finally diethylether.

6.10.2°. Cleavage and Deprotection

By way of example but not limitation, cleavage and deprotection can becarried out as follows: The air-dried peptide resin is treated withethylmethyl-sulfide (EtSMe), ethanedithiol (EDT), and thioanisole(PhSMe) for approximately 20 min. prior to addition of 95% aqueoustrifluoracetic acid (TFA). A total volume of approximately 50 ml ofthese reagents are used per gram of peptide-resin. The following ratiois used: TFA:EtSMe:EDT:PhSme (10:0.5:0.5:0.5). The mixture is stirredfor 3 h at room temperature under an atmosphere of N₂. The mixture isfiltered and the resin washed with TFA (2×3 ml). The combined filtrateis evaporated in vacuo, and anhydrous diethyl ether added to theyellow/orange residue. The resulting white precipitate is isolated byfiltration. See King et al., 1990, Int. J. Peptide Protein Res.36:255–266 regarding various cleavage methods.

6.10.3. Purification of the Peptides

Purification of the synthesized peptides can be carried out by standardmethods including chromatography (e.g., ion exchange, affinity, andsizing column chromatography, high performance liquid chromatography(HPLC)), centrifugation, differential solubility, or by any otherstandard technique.

6.10.4. Conjugation of Peptides to Other Molecules

The peptides of the present invention may be linked to other molecules(e.g., a detectable label, a molecule facilitating adsorption to a solidsubstratum, or a toxin, according to various embodiments of theinvention) by methods that are well known in the art. Such methodsinclude the use of homobifunctional and heterobifunctional cross-linkingmolecules.

The homobifunctional molecules have at least two reactive functionalgroups, which are the same. The reactive functional groups on ahomobifunctional molecule include, for example, aldehyde groups andactive ester groups. Homobifunctional molecules having aldehyde groupsinclude, for example, glutaraldehyde and subaraldehyde. The use ofglutaraldehyde as a cross-linking agent was disclosed by Poznansky etal., 1984, Science 223:1304–1306.

Homobifunctional molecules having at least two active ester unitsinclude esters of dicarboxylic acids and N-hydroxysuccinimide. Someexamples of such N-succinimidyl esters include disuccinimidyl suberateand dithio-bis-(succinimidyl propionate), and their soluble bis-sulfonicacid and bis-sulfonate salts such as their sodium and potassium salts.These homobifunctional reagents are available from Pierce, Rockford,Ill.

The heterobifunctional molecules have at least two different reactivegroups. Some examples of heterobifunctional reagents containing reactivedisulfide bonds include N-succinimidyl 3-(2-pyridyl-dithio)propionate(Carlsson et al., 1978, Biochem J. 173:723–737), sodiumS-4-succinimidyloxycarbonyl-alpha-methylbenzylthiosulfate, and4-succinimidyloxycarbonyl-alpha-methyl-(2-pyridyldithio)toluene.N-succinimidyl 3-(2-pyridyldithio)propionate is preferred. Some examplesof heterobifunctional reagents comprising reactive groups having adouble bond that reacts with a thiol group include succinimidyl4-(N-maleimidomethyl)cyclohexahe-1-carboxylate and succinimidylm-maleimidobenzoate.

Other heterobifunctional molecules include succinimidyl3-(maleimido)propionate, sulfosuccinimidyl4-(p-maleimido-phenyl)butyrate, sulfosuccinimidyl4-(N-maleimidomethyl-cyclohexane)-1-carboxylate,maleimidobenzoyl-N-hydroxy-succinimide ester. The sodium sulfonate saltof succinimidyl m-maleimidobenzoate is preferred. Many of theabove-mentioned heterobifunctional reagents and their sulfonate saltsare available from Pierce.

Additional information regarding how to make and use these as well asother polyfunctional reagents may be obtained from the followingpublications or others available in the art: Carlsson et al., 1978,Biochem. J. 173:723–737; Cumber et al., 1985, Methods in Enzymology112:207–224; Jue et al., 1978, Biochem 17:5399–5405; Sun et al., 1974,Biochem. 13:2334–2340; Blattler et al., 1985, Biochem. 24:1517–152; Liuet al., 1979, Biochem. 18:690–697; Youle and Neville, 1980, Proc. Natl.Acad. Sci. USA 77:5483–5486; Lerner et al., 1981, Proc. Natl. Acad. Sci.USA 78:3403–3407; Jung and Moroi, 1983, Biochem. Biophys. Acta 761:162;Caulfield et al., 1984, Biochem. 81:7772–7776; Staros, 1982, Biochem.21:3950–3955; Yoshitake et al., 1979, Eur. J. Biochem. 101:395–399;Yoshitake et al., 1982, J. Biochem. 92:1413–1424; Pilch and Czech, 1979,J. Biol. Chem. 254:3375–3381; Novick et al., 1987, J. Biol. Chem.262:8483–8487; Lomant and Fairbanks, 1976, J. Mol. Biol. 104:243–261;Hamada and Tsuruo, 1987, Anal. Biochem. 160:483–488; Hashida et al.,1984, J. Applied Biochem. 6:56–63.

Additionally, methods of cross-linking are reviewed by Means and Feeney,1990, Bioconjugate Chem. 1:2–12.

6.10.4.1. Biotinylation of Peptides

Methods of biotinylating peptides are well known in the art. Anyconvenient method may be employed in the practice of the invention. Forexample, the following procedure was used. Ten micrograms of peptide wasdissolved in 100 μl of 0.1% acetic acid. PBS (900 μl) and 3.3 mg ofbiotin-LC-NHS (Pierce, Rockford, Ill.) was added. Following incubationfor 30 minutes at room temperature the biotinylated peptides werepurified over a Superose 12 column (Pharmacia, Piscataway, N.J.).

6.10.5. Synthetic Peptides

Tables 19, 20 and 21 provide the primary structure for various syntheticpeptides manufactured in the practice of the present invention.

TABLE 19 SEQ ID Peptide NO. name Sequence 266 ELAN005 H₂N-C-K(dns)-FITKALGISYGRKKRRQRRRPPQGSQTHQVSLSKQ-CONH₂ 267 ELAN006Ac-CLNGGVKMYVESVDRYVC-CONH₂ 268 FITC- Ac-CLNGGVK(FITC)MYVESVDRYVC-CONH₂ELAN006 269 ELAN006ii H₂N-C-K(dns)-RLNGGVSMYVESVDRYVCR-CONH₂ 167 ELAN007H₂N-RIAGLPWYRCRTVAFETGMQNTQLCSTIVQLSFTPEE- COOH 193 ELAN007iiH₂N-KKRIAGLPWYRCRTVAFETGMQNTQLCSTIVQLSFTPEE- CONH₂ 270 bZElan008biotin-K(dns)SARDSGPAEDGSRAVRLNGVENANTRKSSR (P31) SNPRGRRHP-COOH 271bZElan009 biotin-K(dns)SSADAEKCAGSLLWWGRQNNSGCGSPTKKHLKHRNRSQTSSSSHG-COOH 168 ELAN010H₂N-REFAERRLWGCDDLSWRLDAEGCGPTPSNRAVKHRKPRPR SPAL-COOH 272 bZElan010biotin-K(dns)REFAERRLWGCDDLSWRLDAEGCGPTPSNR AVKHRKPRPRSPAL-COOH 169ELAN012 H₂N- SGSHSGGMNRAYGDVFRELRDRWYATSHHTRPTPQLPRGPN- COOH 273bELAN012 biotin- SGSHSGGMNRAYGDVFRELRDRWYATSHHTRPTPQLPRGPN- COOH 274ZElan012 H₂N- K (dns) SGSHSGGI4NRAYGDVFRELRDRWYATSHHTRPTPQLP RGPN-COOH249 ELAN013 H₂N- SGSPPCGGSWGRFMQGGLFGGRTDGCGAHRNRTSASLEPPSSD Y-CONH₂ 250ELAN014 H₂N- SHSGGMNRAYGDVFRELRDRWNATSHHTRPTPQLPRGPNS- CONH₂ 275bZElan014 biotin- K(dns)SHSGGMNRAYGDVFRELRDRWNATSHHTRPTPQLPRG PNS-CONH₂276 ZElan014 H₂N- K(dns)SHSGGMNRAYGDVFRELRDRWNATSHHTRPTPQLPRG PNS-CONH₂277 ZElan015 H₂N- (DCX11) K(dns)SQGSKQCMQYRTGRLTVGSEYGCGMNPARHATPAYPARLLPRYR-CONH₂ 278 ZElan016 H₂N- (SNi10)K(dns)RVGQCTDSDVRRPWARSCAHQGCGAGTRNSHGCITRP LRQASAH-CONH₂ 279 bZElan017biotin-K(dns)SGSGRVGQCTDSDVRRPWARSCA-CONH₂ 280 ZElan017H₂N-K(dns)RVGQCTDSDVRRPWARSCA-CONH₂ ZElan018 H₂N- 281 (PAX2)K(dns)STPPSREAYSRPYSVDSDSDTNAKHSSHNRRLRTRSR PNG-CONH₂ 282 ZElan019 H₂N-(5PAX5) K(dns)RGSTGTAGGERSGVLNLHTRDNASGSGFKPWYPSNRG HK-CONH₂ 283ZElan020 H₂N-K(dns)SGSGLYANPGMYSRLHSPA-CONH₂ (CY09) 284 bZElan020biotin-K(dns)SGSGLYANPGMYSRLHSPA-CONH₂ (CY09) 285 ZElan021 H₂N- (HAX42)K(dns)SDHALGTNLRSDNAKEPGDYNCCGNGNSTGRKVFNRR RPSAIPT-CONH₂ 286 ZElan022H₂N- (SNi34) K (dns) SPCGGSWGRFMQGGLFGGRTDGCGAHRNRTSASLEPP SSDY-CONH₂287 ZElan023 H₂N- (DCX8) K(dns)RYKHDIGCDAGVDKKSSSVRGGCGAHSSPPRAGRGPRGTMVSRL-CONH₂ 288 ZElan024 H₂N- (P31)K(dns)SARDSGPAEDGSRAVRLNGVENANTRKSSRSNPRGRR HPGG-CONH₂ 289 ZElan025 H₂N-(DAB10) K(dns)SKSGEGGDSSRGETGWARVRSHAMTAGRFRWYNQLPS DR-CONH₂ 290ZElan026 H₂N- (PAX2/ K(dns)SEANLDGRKSRYSSPRRNSSTRPRTSPNSVHARYPSTcontrol) DHD-CONH₂ 291 bELANO27 biotin- (PAX2)SGSGSTPPSREAYSRPYSVDSDSDTNAKHSSHNRRLRTRSRPN G-CONH₂ 251 18C21H₂N-DTNAKHSSHNRRLRTRSRPNG-CONH₂ 292 Fmoc-Fmoc-K(dns)RVGQCTDSDVRRPWARSCAHQG-COOH Z16N23 252 16C23H₂N-CGAGTRNSHGCITRPLRQASAHG-CONH₂ 293 Z16C23H₂N-K(dns)CGAGTRNSHGCITRPLRQASAHG-CONH₂ 294 ZElan028H₂N-K(dns)ENANTRKSSRSNPRGRRHPG-CONH₂ (P31 fragment) 295 ZElan029H₂N-K(dns)TRKSSRSNPRG-CONH₂ (P31 fragment) 296 ZElan030H₂N-K(dns)ENANTRKSSRSNPRG-CONH₂ (P31 fragment) 297 ZElan031H₂N-K(dns)TRKSSRSNPRGRRHPG-CONH₂ (P31 fragment) 298 ZElan032H₂N-K(dns)TNAKHSSHNRRLRTRSRPN-CONH₂ (PAX2 fragment) 299 ZElan033H₂N-K(dns)TNAKHSSHNRRLRTR-CONH₂ (PAX2 fragment) 300 ZElan034H₂N-K(dns)SSHNRRLRTRSRPN-CONH₂ (PAX2 fragment) 301 ZElan035H₂N-K(dns)SSHNRRLRTR-CONH₂ (PAX2 fragment) 302 ZElan036H₂N-K(dns)VRRPWARSCAHQGCGAGTRNS-CONH₂ (SNi10 fragment) 303 ZElan037H₂N-K(dns)CTDSDVRRPWARSC-CONH₂ (SNi10 fragment) 304 ZElan038 H₂N- (PAX2/K(dns)SRANTDGRKSRYSSPRRNSSTEPRLSPNSVHARYPST control) DHD-CONH₂ 1923ZElan039 H₂N-K(dns)ENANTRKSSR-CONH₂ 05 (P31 fragment) 306 ZElan040H₂N-K(dns)SNPRGRRHPG-CONH₂ (P31 fragment) 307 ZElan041H₂N-K(dns)ENANT-CONH₂ (P31 fragment) 308 ZElan042 H₂N-K(dns)ANTRKS-CONH₂(P31 fragment) 309 ZElan043 H₂N-K(dns)TRKSS-CONH₂ (P31 fragment) 310ZElan044 H₂N-K(dns)RKSSR-CONH₂ (P31 fragment) 311 ZElan045H₂N-K(dns)KSSRSN-CONH₂ (P31 fragment) 312 ZElan046H₂N-K(dns)SSRSNPG-CONH₂ (P31 fragment) 313 ZElan047H₂N-K(dns)RSNPRG-CONH₂ (P31 fragment) 314 ZElan048 H₂N-K(dns)SNPRG-CONH₂(P31 fragment) 315 ZElan049 H₂N-K(dns)PRGRRH-CONH₂ (P31 fragment) 316ZElan050 H₂N-K(dns)RRHPG-CONH₂ (P31 fragment) 317 ZElan051H₂N-K(dns)KSSRGN-CONH₂ (HepC) 318 ZElan052H₂N-K(dns)KTSERSQPRGRRQPG-CONH₂ (HepC) ZElan053H₂N-K(dns)TrKSSrSNPrGrrHPG-CONH₂ 319 (P31 analog) 320 ZElan054H₂N-K(dns)TRKSSrSNPRGrRHPG-CONH₂ (P31 analog) 321 ZElan055H₂N-K(dns)TNAKHSSHN-CONH₂ (PAX2 fragment) 322 ZElan056H₂N-K(dns)RRLRTRSRPN-CONH₂ (PAX2 fragment) 323 ZElan057H₂N-K(dns)RRLRTRSR-CONH₂ (PAX2 fragment) 324 ZElan058H₂N-K(dns)RRLRTR-CONH₂ (PAX2 fragment) 325 ZElan059H₂N-K(dns)rrLrTrSrPN-CONH₂ (PAX2 analog) 326 ZElan06OH₂N-K(dns)SDHALGTNLRSDNAKEPGDYNCCGNG-CONH₂ (HAX42 fragment) 327 ZElan061H₂N-K(dns)GDYNCCGNGNSTGRKVFNRRRPSAIPT-CONH₂ (HAX42 fragment) 328ZElan062 H₂N-K(dns)SDHALGTNLRSDNAKEPG-CONH₂ (HAX42 fragment) 329ZElan063 H₂N-K(dns)GDYNCCGNGNSTG-CONH₂ (HAX42 fragment) 330 ZElan064H₂N-K(dns)RKVFNRRRPSAIPT-CONH₂ (HAX42 fragment) 331 ZElan065H₂N-K(dns)RKVFNRRRPS-CONH₂ (HAX42 fragment) 332 ZElan066H₂N-K(dns)NRRRPSAIPT-CONH₂ (HAX42 fragment) 333 ZElan067H₂N-K(dns)NRRRPS-CONH₂ (HAX42 fragment) 334 Elan018 H₂N- (PAX2 noSTPPSREAYSRPYSVDSDSDTNAKMSSHNRRLRTRSRPNG- dns) CONH₂ 335 Elan021H₂N-SDHALGTNLRSDNAKEPGDYNCCGNGNSTGRKVFNRRRPS (HAX42 no AIPT-CONH₂ due)336 ZElan070 H₂N-K(dns)SDHALGTNLRSDNAKEPGDYNCCGNGNST- (HAX42 CONH₂fragment) 337 ZElan071 H₂N-K(dns)NLRSDNAKEPGDYNCCGNGNSTGRKVFNR- (HAX42CONH₂ fragment) 338 ZElan072 H₂N-K(dns)PGDYNCCGNGNSTGRKVFNRRPSAIPT-CONH₂(HAX42 fragment) 339 ZElan073 H₂N-K(dns)ASHNRRLRTR-CONH₂ (PAX2 fragment)340 ZElan074 H₂N-K(dns)SAHNRRLRTR-CONH₂ (PAX2 fragment) 341 ZElan075H₂N-K(dns)SSANRRLRTR-CONH₂ (PAX2 fragment) 342 ZElan076H₂N-K(dns)SSHARRLRTR-CONH₂ (PAX2 fragment) 343 ZElan077H₂N-K(dns)SSHNARLRTR-CONH₂ (PAX2 fragment) 344 ZElan078H₂N-K(dns)SSHNRALRTR-CONH₂ (PAX2 fragment) 345 ZElan079H₂N-K(dns)SSHNRRARTR-CONH₂ (PAX2 fragment) 346 ZElan080H₂N-K(dns)SSHNRRLATR-CONH₂ (PAX2 fragment) 347 ZElan081H₂N-K(dns)SSHNRRLRAR-CONH₂ (PAX2 fragment) 348 ZElan082H₂N-K(dns)SSHNRRLRTA-CONH₂ (PAX2 fragment) 349 Elan035H₂N-SSHNRRLRTR-CONH₂ 350 ZElan083 H₂N- (PAX2/ K (dns)GRNHDVVSSNTHKSYRSPRSASYPRLSNDRTDRTEPA control) PSS-CONH₂ 351 ZElan084H₂N-K(dns)RNTRNKTSRLSANPHRSHR-CONH₂ (PAX2/ control) 352 Elan032ZH₂N-TNAKHSSHNRRLRTRSRPN K(dns)-CONH₂ (PAX2 fragment) 353 Elan057ZH₂N-RRLRTRSRK(dns)-CONH₂ (PAX2 fragment)

TABLE 20 SEQ. ID. Name Description Sequence No. ZElan087 HAX42-1 (20mer) H₂N—K(dns)SDHALGTNLRSDNAKEPGDY 354 ZElan088 HAX42-2 (20 mer)H₂N—K(dns)SDNAKEPGDYNCCGNGNSTG 355 ZElan089 HAX42-3 (15 mer)H₂N—K(dns)SDHALGTNLRSDNAK 356 ZElan090 HAX42-4 (15 mer)H₂N—K(dns)EPGDYNCCGNGNSTG 357 ZElan091 HAX42-5 (14 mer)H₂N—K(dns)PGDYNCCGNGNSTG 358 ZElan092 HAX42-6 (10 mer)H₂N—K(dns)PGDYNCCGNG 359 ZElan093 HAX42-7 (10 mer) H₂N—K(dns)NCCGNGNSTG360 ZElan100 P31 16 mercyclic

361 ZElan101 P31 16 mercyclic D form

362 ZElan103 PAX2 15 mercyclic

363 ZElan103A PAX2 15 mercyclic(internal)

364 ZElan104 PAX2 15 mercyclic(internal)

365 ZElan105 PAX2 Ala Scan 1 H₂N—K(dns)ANAKHSSHNRRLRTR 366 ZElan106 PAX2Ala Scan 2 H₂N—K(dns)TAAKNSSHNRRLRTR 367 ZElan107 PAX2 Ala Scan 3H₂N—K(dns)TNGKNSSHNRRLRTR 368 ZElan108 PAX2 Ala Scan 4H₂N—K(dns)TNAAHSSHNRRLRTR 369 ZElan109 PAX2 Ala Scan 5H₂N—K(dns)TNAKASSHNRRLRTR 370 ZElan110 PAX2 Ala Scan 6H₂N—K(dns)TNAKHASHNRRLRTR 371 ZElan111 PAX2 Ala Scan 7H₂N—K(dns)TNAKHSAHNRRLRTR 372 ZElan112 PAX2 Ala Scan 8H₂N—K(dns)TNAKHSSANRRLRTR 373 ZElan113 PAX2 Ala Scan 9H₂N—K(dns)TNAKHSSHARRLRTR 374 ZElan114 PAX2 Ala Scan 10H₂N—K(dns)TNAKHSSHNARLRTR 375 ZElan115 PAX2 Ala Scan 11H₂N—K(dns)TNAKHSSHNRALRTR 376 ZElan116 PAX2 Ala Scan 12H₂N—K(dns)TNAKHSSHNRRARTR 377 ZElan117 PAX2 Ala Scan 13H₂N—K(dns)TNAKHSSHNRRLATR 378 ZElan118 PAX2 Ala Scan 14H₂N—K(dns)TNAKHSSHNRRLRAR 379 ZElan119 PAX2 Ala Scan 15H₂N—K(dns)TNAKHSSHNRRLRTA 380 ZElan123 PAX2 15 merH₂N—K(dns)Lys-TNAKHSSHNrrLrTr 381 cyclic D form ZElan124 PAX2 15 mer DH₂N—K(dns)TNAKHSSHNrrLrTr 382 form ZElan125 PAX2 10 mercyclic

383 ZElan126 PAX2 10 mercyclic D form

384 ZElan127 PAX2 10 mercyclic

385 ZElan128 PAX2 10 mercyclic D form

386 ZElan129 PAX2 15 mer H₂N—K(dns)TNAKHSSHNRRLRTR 387 ZElan130 HAX42 14mer Ala H₂N—K(dns)AGDYNCCGNGNSTG 388 Scan 1 ZElan131 HAX42 14 mer AlaH₂N—K(dns)PADYNCCGNGNSTG 389 Scan 2 ZElan132 HAX42 14 mer AlaH₂N—K(dns)PGAYNCCGNGNSTG 390 Scan 3 ZElan133 HAX42 14 mer AlaH₂N—K(dns)PGDANCCGNGNSTG 391 Scan 4 ZElan134 HAX42 14 mer AlaH₂N—K(dns)PGDYACCGNGNSTG 392 Scan 5 ZElan135 HAX42 14 mer AlaH₂N—K(dns)PGDYNACGNGNSTG 393 Scan 6 ZElan136 HAX42 14 mer AlaH₂N—K(dns)PGDYNCAGNGNSTG 394 Scan 7 ZElan137 HAX42 14 mer AlaH₂N—K(dns)PGDYNCCANGNSTG 395 Scan 8 ZElan138 HAX42 14 mer AlaH₂N—K(dns)PGDYNCCGAGNSTG 396 Scan 9 ZElan139 HAX42 14 mer AlaH₂N—K(dns)PGDYNCCGNANSTG 397 Scan 10 ZElan140 HAX42 14 mer AlaH₂N—K(dns)PGDYNCCGNGASTG 398 Scan 11 ZElan141 HAX42 14 mer AlaH₂N—K(dns)PGDYNCCGNGNATG 399 Scan 12 ZElan142 HAX42 14 mer AlaH₂N—K(dns)PGDYNCCGNGNSAG 400 Scan 13 ZElan143 HAX42 14 mer AlaH₂N—K(dns)PGDYNCCGNGNSTA 401 Scan 14

GST fusion proteins of GIT peptides are shown in Table 21.

TABLE 21 Source Clone # GST Fusion Sequence SEQ ID NO. DCX11 98gst-SQGSKQCMQYRTGRLTVGSEYGCGMNPARHATPAYPARLLPRYR 213 HAX42 99gst-SDHALGTNLRSDNAKEPGDYNCCGNGNSTGRKVFNRRRPSAIPT 214 SNi34 100gst-SPCGGGSWGRFMQGGLFGGRTDGCGAHRNRTSASLEPPSSDY 215 5PAX5 97gst-RGSTGTAGGERSGVLNLHTRDNASGSGFKPWYPSNRGHK 216 SNi28 84gst-SHSGGMNRAYGDVFRELRDRWNATSHHTRPTPQLPRGPN 217 SNi28 85 gst-SHSGGNNRAY218 SNi28 86 gst-GDVFRELRDR 219 SNi28 87 gst-WNATSHHTRP 220 SNi28 88gst-TPQLPRGPN 221 SNi28 89 gst-GDVFRELRDRWNATSHHTRP 222 SNi28 90gst-WNATSHHTRPTPQLPRGPN 223 SNi28 91 gst-GDVFRELRDRWNATSHHTRPTPQLPRGPN224 SNi28 92 gst-SHSGGMNRAYGDVFRELRDRWNATSAATRPTPQLPRGPN 225 P31 93gst-SARDSGPAEDGSRAVRLNGVENANTRKSSRSNPRGRRHP 226 P31 101gst-SARDSGPAEDGSRAVRLNG 227 P31 102 gst-DGSRAVRLNGVENANTRKSSR 228 P31103 gst-ENANTRKSSRSNPRGRRHP 229 P31 110 gst-ENANTRKSSR 230 P31 111gst-RKSSRSNPRG P31 112 gst-SNPRGRRHP 232 P31 119 gst-TRKSSRSNPRG 233PAX2 94 gst-STPPSREAYSRPYSVDSDSDTNAKHSSHNRRLRTRSRPN 234 PAX2 104gst-STPPSREAYSRPYSVDSDSD 235 PAX2 105 gst-SRPYSVDSDSDTNAKHSSHNR 236 PAX2106 gst-TNAKHSSHNRRLRTRSRPN 237 PAX2 113 gst-TNAKHSSHN 238 PAX2 114gst-SSHNRRLRTR 239 PAX2 115 gst-RRLRTRSRPN 240 SNi10 96gst-RVGQCTDSDVRRPWARSCAHQGCGAGTRNSHGCITRPLRQASAH 241 SNi10 116gst-RVGQCTDSDVRRPWARSCA 242 SNi10 117 gSt-VRRPWARSCAHQGCGAGTRNS 243SNi10 118 gst-GTRNSHGCITRPLRQASAH 244 DCX8 95gst-RYKHDIGCDAGVDKKSSSVRGGCGAHSSPPRAGRGPRGTMVSRL 245 DCX8 107gst-RYKHDIGCDAGVDKKSSSVRGGCG 246 DCX8 108 gst-GCDAGVDKKSSSVRGGCGAHSSPPRA247 DCX8 109 gst-GAHSSPPRAGRGPRGTMVSRL 248

6.10.6. Peptide Stability

The relative stability for ZElan031 (SEQ ID NO:297), ZElan053 (SEQ IDNO:319) and ZElan054 (SEQ ID NO:320) was determined in simulatedintestinal fluid (SIF) SIF was made by dissolving 100 mg of pancreatin(Sigma cat#P-1625, lot# 122H0812)in 8.4 ml of phosphate stock solution,adjusting the pH to 7.5 with 0.2N NaOH and adjusting the volume to 10 mlwith water.

Peptide (3.25 mg) was dissolved in 3.25 ml of 10,000 fold diluted SIFsolution at 37° C. Aliquots (0.7 ml) of the digestion solution were thenwithdrawn at <1 min, 1h, 3h, and 21h or 24h. The samples were quicklypassed through a syringe filter (Millipore Millex-GV 0.22 μm, part#SLGV025LS, lot# H2BM95250) and 300 μL of the filtered solution wasimmediately injected onto a Hewlett-Packard HPLC system equipped with aC-8 column (Applied Biosystems column and guard column: column-p/n0711-0023 Spheri-5 ODS 5 μm, 220×4.6 mm). The products were eluted at1.5 ml/min using an acetonitrile-water gradient. The major fluorescentpeaks were collected, lyopholized and identified by MS analysis.

The HPLC gradient used was:

Time (min) Solvent Mixture 0 95% H₂0–5% acetonitrile (0.1% TFA) 5 95%H₂0–5% acetonitrile (0.1% TFA) 35 85% H₂0–15% acetonitrile (0.1% TFA)linear solvent change 40 0% H₂0–100% acetonitrile (0.1% TFA) linearsolvent change 45 95% H₂0–5% acetonitrile (0.1% TFA) linear solventchange 52 95% H₂0–5% acetonitrile (0.1% TFA) linear solvent change

As shown in Table 22, the relative stability (to SIF) for the threepeptides was found to be ZElan053>ZElan054>ZElan031 (SEQ IDNOs:319,320,297, respectively). Enzymatic cleavage of the peptide wasfound to occur at arginine and/or lysine as expected. The replacement of1-amino acids with their D-amino acid analogs significantly reduced therate of proteolysis at these residues.

TABLE 22 Percent Remaining at: Peptide 1 m 1 h 3 h 24 h Rel. Stab. SEQID NO ZElan031 100 38.7 0 0 3 297 ZElan054 97.4 58.2 11.6 2.7 2 320ZElan053 100 98.3 98.1 94.0 1 3217. Characterization of Peptide-Coated Particles

Binding of Peptide-Coated PLGA Nanoparticles to Fixed Caco-2 Cells

Binding of nanoparticles coated with targeting peptides to fixed Caco-2cells was investigated using an ELISA assay based on reaction ofantibody with the dansyl moiety present on the peptides. Isoelectricpoints of selected synthetic peptides are shown in Table 23(corresponding SEQ ID NOS. are shown in Table 7). Correspondingdansylated synthetic GIT binding peptides are given in Table 24.

TABLE 23 SEQ ID Peptide Sequence pI NO. P31SARDSGPAEDGSRAVRLNGVENANTRKSSRSNPRGRRHP 12.26 43 5PAX5RGSTGTAGGERSGVLNLHTRDNASGSGFKPWYPSNRGHK 11.49 46 SNi10RVGQCTDSDVRRPWARSCAHQGCGAGTRNSHGCITRPLRQASAH 10.45 4 SNi34SPCGGSWGRFMQGGLFGGRTDGCGAHRNRTSASLEPPSSDY 8.25 6 DCX11SQGSKQCMQYRTGRLTVGSEYGCGMNPARHATPAYPARLLPRYR 10.44 24 DCX8RYKHDIGCDAGVDKKSSSVRGGCGAHSSPPRAGRGPRGTMVSRL 11.03 23 HAX42SDHALGTNLRSDNAKEPGDYNCCGNGNSTGRKVFNRRRPSAIPT 9.62 52 PAX2STPPSREAYSRPYSVDSDSDTNAKHSSHNRRLRTRSRPN 11.26 55

TABLE 24 SEQ ID Peptide Sequence NO. P31H₂N-K(dns)SARDSGPAEDGSRAVRLNGVENANTRKSSRSNPRGRRHPGG-CONH₂ 288 5PAX5H₂N-K(dns)RGSTGTAGGERSGVLNLHTRDNASGSGFKPWYPSNRGHK-CONH₂ 282 SNi10H₂N-K(dns)RVGQCTDSDVRRPWARSCAHQGCGAGTRNSHGCITRPLRQASAH-CONH₂ 278 SNi34H₂N-K(dns)SPCGGSWGRFMQGGLFGGRTDGCGAHRNRTSASLEPPSSDY-CONH₂ 286 DCX11H₂N-K(dns)SQGSKQCMQYRTGRLTVGSEYGCGMNPARHATPAYPARLLPRYR-CONH₂ 277 DCX8H₂N-K(dns)RYKHDIGCDAGVDKKSSSVRGGCGAHSSPPRAGRGPRGTMVSRL-CONH₂ 287 HAX42H₂N-K(dns)SDHALGTNLRSDNAKEPGDYNCCGNGNSTGRKVFNRRRPSAIPT-CONH₂ 285 PAX2H₂N-K(dns)STPPSREAYSRPYSVDSDSDTNAKHSSHNRRLRTRSRPNG-CONH₂ 281 DAB10H₂N-K(dns)SKSGEGGDSSRGETGWARVRSHAMTAGRFRWYNQLPSDR-CONH₂ 289Method:

Confluent Caco-2 monolayers grown in 96-well plates (p38) were fixed andtreated with 0.1% phenylhydrazine before blocking with 0.1% BSA in PBS.Control and dansyl peptide-coated nanoparticles were resuspended insterile water at 10 mg/ml and stirred with a magnet for 1 h at roomtemperature. Samples consisted of: (1) blank nanoparticle control, (2)scrambled PAX2-coated nanoparticles, (3) PAX2-coated nanoparticles, (4)HAX42-coated nanoparticles, (5) PAX2/HAX42-coated nanoparticles, and (6)8 peptide-coated nanoparticles.

Nanoparticles were added to the cells at 10 mg/ml in 100 μl 1% BSA-PBS(no Tween80 is used in this assay) and 2-fold serially-diluted. The96-well plates were incubated for 1 h at room temperature. The plateswere washed 5 times with 1% BSA-PBS and 100 μl of anti-dansyl antibody(Cytogen DB3-226.3; 0.5 μg/ml; batch May 1997) was added per well andthe plates incubated 1 h at room temperature. The wells were washed 5times with 1% BSA-PBS; 1001 of goat anti-mouse λ:HRP antibody (SouthernBiotechnology CN. 1060-05; 1:10,000) was added per well, and the platesincubated 1 h at room temperature. After washing 5 times with 1%BSA-PBS, 100 μl of MB peroxidase substrate (KPL CN. 50-76-00) was addedto the wells and the optical density at 650 nm was measured after 15minutes.

As shown in FIGS. 13A–B, a decreasing anti-dansyl ELISA response wasobserved for nanoparticles coated with PAX2, HAX2, PAX2+HAX2, and amixture of 8 targeting peptides, when decreasing amounts of thenanoparticles were applied to fixed Caco-2 cells. No concentrationeffect was observed for blank nanoparticles or nanoparticles coated witha scrambled version of PAX2 peptide. Nanoparticles coated with PAX2,HAX2, PAX2+HAX2, and the 8 peptide mix, showed increased responserelative to blank nanoparticles or nanoparticles coated with a scrambledversion of PAX2 peptide. The OD values were low relative to thosenormally observed for GST-peptide fusion binding to fixed Caco-2 cells.

Table 25 below shows the insulin potency and level of peptides coatedonto the particles (measured by fluorescense) for formulation 1particles (formulation by the coacervation method given below).

TABLE 25 ELISA of dansylated peptides and insulin coated PLGA particlesBlend Insulin Peptide Peptide mg/g μl/mg PAX2 60.7 3.51 HAX42 55.9 2.93PAX2 SCRAMBLED 57.7 1.26 P31 67.0 1.22 5PAX5 52.7 2.83 SNi10 59.5 1.75SNi34 61.5 4.03 DCX8 59.1 1.87 DAB10 55.9 1.99

The standard ELISA procedure was modified as follows. Peptides andparticles were diluted to an appropriate concentration in PBS containing1% BSA (particles were sonicated to achieve a homogeneous solution),titered and incubated one hour at room temperature. Following fivewashes with PBS containing 1% BSA, an in-house IgG1λ anti-dansylmonoclonal antibody was added (diluted to 1 μg/ml in 1% BSA-PBS) and theplates were incubated for one hour. After five more washes goatanti-mouse λ-HRP was added (Southern Biotechnology Associates Inc.,Birmingham, Ala., diluted 1:10,000 in 1% BSA-PBS) and the plates wereincubated one hour. After five washes, plates were developed with TMBperoxidase substrate (Kirkegard and Perry, Gaithersburg, Md.). All datais presented with background binding subtracted. Tween 20 was not addedto the diluent or the washes when insulin coated PLGA particles wereincluded in the assay.

FIGS. 14A–14B show the binding of the dansylated peptide SNi10 to hSIand BSA.

8. Binding of Synthetic Peptides and Peptide-Coated Particles to S100and P100 Fractions Derived from Caco-2 Cells

8.1. Detection of Binding to Membrane (P100) and Cytosolic (S100)Fractions

Caco-2 cell membrane (P100) and cytosolic (S100) fractions were preparedusing a modification of the method described in Kinsella, B. T.,O'Mahony, D. J. and G. A. FitzGerald, 1994, J. Biol. Chem. 269(47):29914–29919. Confluent Caco-2 cell monolayers (grown in 75 cm² flasksfor up to 1 week at 37° C. and 5% CO₂) were washed twice in Dulbecco'sPBS (DPBS) and the cells were harvested by centrifugation at 1000 rpmafter treatment with 10 mM EDTA-DPBS. The cells were washed 3 times inDPBS and the final cell pellet was resuspended in 3 volumes of ice coldHED buffer (20 mM HEPES (pH 7.67), 1 mM EGTA, 0.5 mM dithiothreitol, 1mM phenylmethylsulphonyl fluoride (PMSF)). The cells were allowed toswell for 5 min on ice prior to homogenization for 30 sec. Thehomogenates were centrifuged at 40,000 rpm for 45 min at 4° C. Thesupernatant (S100) was removed and the pellet (P100) was resuspended inHEDG buffer (20 mM HEPES (pH 7.67), 1 mM EGTA, 0.5 mM dithiothreitol,100 mM NaCl, 10% glycerol, 1 mM PMSF). Protein concentrations weredetermined using the Bradford assay (Bradford, M. M., 1976, Anal.Biochem. 72: 248–254).

Binding of peptide and/or peptide-coated PLGA particles to membrane(P100) and cytosolic (S100) fractions was assessed by detection of thedansyl moiety incorporated in the peptide. Costar ninety six well ELISAplates were coated with S100 and P100 fractions (100 μg/ml in 0.05 MNaHCO₃) overnight at 4° C. The plates were blocked with 0.5% bovineserum albumin in DPBS for 1 h at room temperature and washed 3 times in1% BSA-DPBS. Peptide-coated particles or peptides were dispersed in thesame buffer and added to the plates at concentrations in the range0.0325–0.5 mg/well. After 1 h at room temperature the plates were washed5 times in 1% BSA-DPBS and 100 μl of anti-dansyl antibody (CytogenDB3-226.3; 0.5 μg/ml) was added per well. The plates were incubated for1 h at room temperature. The wells were washed 3 times in 1% BSA-DPBSand 100 μl of goat anti-mouse IgGλ:HRP antibody (Southern Biotechnology1060-05; 1:10,000) was added per well. The plates were incubated for 1 hat room temperature. After washing 3 times in 1% BSA-DPBS 100 μl of TMBsubstrate (3,3′,5′,5-tetramethylbenzidine; Microwell PeroxidaseSubstrate System (Kirkegaard and Perry Laboratories 50-76-00)) was addedand the optical density was measured at 650 nm at various timeintervals.

8.2. Binding of Peptide-Coated PLGA Particles

A novel assay system is provided by the instant invention for detectionof binding of peptide-coated PLGA particles to membrane (P100) andcytosolic (S100) fractions derived from live Caco-2 cells. Theabsorbance readings obtained using this assay system were substantiallyhigher than those obtained using similar peptide-coated PLGA particleconcentrations on fixed Caco-2 cells. This greater sensitivity togetherwith the derivation of the S100 and P100 fractions from live Caco-2cells suggests that this assay may be the assay system of choice fordetection of peptide-coated PLGA particle binding. The assay wasconcentration dependent and peptide/particle correlation permitteddifferentiation between specific and non-specific binding interactions.

Binding of peptide-coated PLGA particles was assessed using S100 andP100 fractions derived from live Caco-2 cells as described above. Thefractions were coated onto 96-well plates at 10 μg/well in 0.05 M NaHCO₃and peptide-coated PLGA particles were assayed by ELISA atconcentrations in the range 0.0325–0.5 mg/well.

FIGS. 15A and 15B illustrate the data obtained on S100 and P100fractions respectively for particles coated with no peptide, scrambledPAX2 (control), P31 D-Arg 16-mer (ZElan053, SEQ ID NO: 319), HAX42, PAX2and HAX42/PAX2. Using particle concentrations of 0.0325–0.5 mg/well alltest peptide-coated PLGA particles exhibited greater binding to both theS100 and P100 fractions than the scrambled PAX2 coated controlparticles. All particles except P31 D-Arg 16-mer (ZElan053, SEQ ID NO:319) exhibited greater binding to the P100 fraction than the S100fraction. Greater binding of the P31 D-Arg 16-mer (ZElan053, SEQ ID NO:319) coated particles to the S100 fraction may be indicative ofnon-specific binding due to the D-Arg modification of the P31 peptide(SEQ ID NO:270).

Binding of PLGA particles coated with varying concentrations of PAX2peptide ranging from 0.05–5.0 mg/g was assessed using a) fixed Caco-2cells (P35) and b) S100 and P100 fractions (Caco-2 P33). The particleswere assayed at concentrations in the range 0.03125–0.0625 mg/well.

Using a particle concentration of 0.0625 mg/well, all PAX2 coatedparticles except those coated at 0.05 mg/g exhibited greater binding tofixed Caco-2 cells than the scrambled PAX2 coated control particles.There appeared to be a concentration effect with increasing PAX2 peptideconcentration resulting in improved Caco-2 cell binding (in the range0.05–1.0 mg/g). However all absorbance readings were low and binding ofthe PAX2 (5 mg/g) was not consistent with this pattern.

Using particle concentrations of 0.03125–0.0625 mg/well all test peptidecoated particles except PAX2 (0.05 mg/g) exhibited comparable or greaterbinding to both the S100 and P100 fractions than the scrambled PAX2coated control particles. All particles exhibited greater binding to theP100 fraction than the S100 fraction. Binding to both the S100 and P100fractions was directly proportional to the concentration of the PAX2peptide on the particle. The absorbance readings obtained using thisassay system were substantially higher than those obtained on the fixedCaco-2 cells.

The effect of blocking solution on binding of peptide-coated PLGAparticles to P100 fractions (Caco-2 P35) was assessed using 1% bovineserum albumin (BSA) and 1% milk powder blocking solutions to assessbackground binding. The following particles were assayed atconcentrations in the range 0.03125–0.0625 mg/well: no peptide;scrambled PAX2; and a range of PAX2 coated particles having peptideconcentrations from 5–0.05 mg/g. As previously observed using 1% BSA,all test peptide coated particles except PAX2 coated at 0.05 mg/gexhibited comparable or greater binding to the P100 fractions than thescrambled PAX2 coated control particles. Binding to P100 fractions wasdirectly proportional to the concentration of the PAX2 peptide on theparticle (although in this instance PAX2 (5 mg/g) exhibited slightlylower binding than PAX2 (1 mg/g)). A similar trend was observed using 1%milk powder and a particle concentration of 0.0625 mg/well. However allabsorbance readings were low when 1% milk powder was used and thebinding pattern was not detectable using particles at a concentration of0.0625 mg/well.

Non-specific binding of peptide-coated PLGA particles to plastic wasalso assessed using 1% BSA and 1% milk powder blocking solutions. Thebinding pattern observed above could be detected when BSA was used;however, absorbance readings were substantially lower and binding ofparticles PAX2 (0.1 and 0.05 mg/g respectively) was not detectable. When1% milk powder was used, all absorbance readings were low and no bindingpattern was detectable. BSA was chosen for blocking in subsequentassays.

8.3. Comparison of Peptide-Coated Particle and Synthetic Peptide Bindingto P100 Fractions

Binding of dansylated peptides to P100 fractions was assessed todetermine if peptide binding was predictive of peptide-coated particlebinding. FIG. 16 illustrates the data obtained for the dansylatedpeptides A) HAX42, P31 D-form and scrambled PAX2 and B) PAX2, HAX42 andscrambled PAX2.

Two consecutive assays produced substantial variations in absorbancereadings. Initially, the HAX42 peptide exhibited strong binding whencompared to the scrambled PAX2 control. The P31 D-form peptide(ZElan053, SEQ ID NO: 319) exhibited binding at the highest dilutiononly. In the repeat assay, HAX42 also exhibited significant bindingcompared to the scrambled PAX2 control. However, the scrambled PAX2control and HAX42 produced relatively high absorbance values compared tothose obtained in the previous assay. The PAX2 peptide wasindistinguishable from the scrambled PAX2 control. Peptide/particlebinding correlation is summarized as follows in Table 26:

TABLE 26 Peptide/particle assay correlation Assay Peptide correlationHAX42 + PAX2 +/− P31 D-form − Scrambled +/− PAX2 + positive; +/−equivocal; − negative

Peptide/particle binding correlated well for the HAX42 peptide. Incontrast, no correlation could be detected for the P31 D-form (ZElan053,SEQ ID NO: 319) peptide. Since the P31 D-form peptide-coated particlesexhibited greater binding to the S100 fraction than the P100 fraction(unlike the other test peptides) it appears that the particle bindinginteraction was non-specific or that some other molecule was competingfor binding to the P100 fraction but not to the S100 fraction. Thus thepeptide/particle assay correlation may be useful for distinguishingbetween specific and non-specific binding interactions. The scrambledPAX2 control produced variable results so that it was difficult toassess the PAX2 binding correlation.

8.4. Determination of HAX42 and PAX2 Binding Motif Sequences

Peptides and GST fusion proteins of HAX42, PAX2 and various derivativeswere assayed using peptide ELISA to P100 membrane fractions derived fromCaco-2 cells. The GST-PAX2 protein and PAX2 peptide data indicate that acore binding motif lies in the amino acid sequence TNAKHSSHNRRLRTR (SEQID NO: 402) otherwise named GST-106 and ZElan033 (SEQ ID NO: 299).Similarly, the HAX42 peptide data suggest that a core binding motif forHAX42 lies in the amino acid sequence PGDYNCCGNCNSTG (SEQ ID NO: 403),otherwise named ZElan091 (SEQ ID NO: 358).

The peptides and proteins were analyzed by a dansylated peptide ELISAmethod in which 96 well plates were coated overnight at 4° C. with 100μl/well coating protein (normally 100 μg/ml P100 membrane fraction) in0.05M carbonate buffer pH9.6. Nonspecific binding was blocked using 200μl/well, 2% Marvel/PBS for 2 hours at 37° C. prior to incubation withdansylated peptides. The plates were washed three times with PBS/0.05%Tween 20 and after each subsequent incubation step. The peptides werediluted in blocking solution at a starting concentration of 100 μg/mland diluted 1:2 downwards, 100 μl/well, followed by incubation at roomtemperature for 1 hour, exactly. A buffer blank control was included toensure that background binding to plastic was not due to the antibodiesused in the assay system. To detect the dansylated peptides, a mouseanti-dansyl antibody (DB3, Cytogen Corp.) at 1:1340 dilution in blockingbuffer and 100 μl/well was added followed by incubation at roomtemperature for 1 hour. The plates were then incubated with ananti-mouse λ-HRP conjugated antibody (Southern Biotech 1060-05) at a1:10,000 dilution in blocking solution, 100 μl/well for 1 hour at roomtemperature. Plates were developed using 75 μl/well Bionostics TMBsubstrate and incubated for approximately 10 minutes. The developingreaction was stopped using Bionostics Red Stop solution (25 μl/well),and the optical density of the plates was read at 650 nm.

GST-PAX2 Peptides—Relative Binding to P100 Fractions

After subtraction of the GST-peptide binding to plastic from P100binding values, the binding of GST-PAX2 peptides were represented as aratio of GST-HAX42 binding to P100, which was given the arbitrary valueof 1.00. The following ratios were determined from binding to P100 ofGST-peptides at a peptide concentration of 20 μg/ml. Bold denotespositive binding to the P100 membrane fraction.

TABLE 27 GST-peptide Value GST-HAX42 1.00 GST-PAX2 1.79 GST-104 0.01GST-105 −0.08 GST-106 2.71 GST-113 0.26 GST-114 0.17 GST-115 0.36 GST0.48

TABLE 28 GST-peptide Amino Acid Sequence SEQ. ID. No. GST-PAX2STPPSREAYSRPYSVDSDSDTNAKHSSHNRRLRTRSRPN  55 GST-104 STPPSREAYSRPYSVDSDSD170 GST-105 STPPSREAYSRPYSVDSDSDTNAKHSSHN 171 GST-106                    TNAKHSSHNRRLRTRSRPN 172 GST-113                    TNAKHSSHN 173 GST-114                         SSHNRRLRTRSRPN 174 GST-115                         RRLRTRSRPN 175

PAX2 Peptides—Relative Binding to P100 Fractions

ZElan021 (SEQ ID NO: 285), full length HAX42, was given the arbitraryvalue of 1.00 for binding to P100 at a given peptide concentrationdetermined from the signal-to-noise ratio data. PAX2 and its derivativesare given as a ratio of HAX42 value to reflect their binding abilitiesto P100 membrane fractions derived from a Caco-2 cell line as shown inTable 329. Table 30 provides a line-up of the PAX2 peptides showing thepositive binding peptides in boldface. The GST-PAX2 peptide and PAX2peptide data agree, demonstrating that a binding motif is in the aminoacid sequence TNAKHSSHNRRLRTR (SEQ ID NO: 402)(GST-106 and ZElan033, SEQID NO: 299).

TABLE 29 Binding value Binding value PAX2 Binding value Binding valueBinding value Binding value at 50 μg/ml at 50 μg/ml SEQ ID NO. peptideat 20 μg/ml at 20 μg/ml at 50 μg/ml at 50 μg/ml (Jackson Ab) (SouthernAb) 281 ZElan018 −0.33 1.07 0.95 1.01 298 ZElan032 1.43 2.87 0.95 1.06299 ZElan033 0.35 1.57 0.80 0.66 301 ZElan035 0.12 0.43 0.81 0.77 321ZElan055 0.99 0.73 1.10 0.59 322 ZElan056 0.00 0.16 0.21 0.21 323ZElan057 0.08 0.56 0.25 324 ZElan058 0.05 0.47 0.16 339 ZElan073 0.07−0.11 0.49 0.66 0.49 340 ZElan074 0.06 0.82 0.52 0.71 0.48 341 ZElan0750.13 0.52 0.38 0.47 0.32 342 ZElan076 0.08 1.00 0.41 0.60 0.42 343ZElan077 0.20 0.76 0.54 0.73 0.52 344 ZElan078 0.11 0.87 0.69 0.68 0.47345 ZElan079 0.31 0.97 0.68 0.83 0.53 346 ZElan080 0.23 0.84 0.45 0.670.38 347 ZElan081 0.01 0.89 0.47 348 ZElan082 0.00 0.92 0.40 350ZElan083 0.43 0.63 1.03 0.88 351 ZElan084 1.06 0.93 1.16 0.77

TABLE 30 Amino acid sequence SEQ ID NO: PAX2 Peptide ZElan018 H₂N-K(dns)STPPSREAYSRPYSVDSDSDTNAKHSSHNRRLRTRSRPNG -CONH ₂ 281 ZElan032                    H ₂N-K(dns)TNAKHSSHNRRLRTRSRPN-CONH₂ 298 ZElan033                    H ₂N-K(dns)TNAKHSSHNRRLRTR-CONH₂ 299 ZElan034                         H₂N-K(dns)SSHNRRLRTRSRPN-CONH₂ 300 ZElan035                         H ₂N-K(dns)SSHNRRLRTR-CONH₂ 301 ZElan055                    H₂N-K(dns)TNAKHSSHN-CONH₂ 321 ZElan056                             H₂N-K(dns)RRLRTRSRPN-CONH₂ 322 ZElan057                             H₂N-K(dns)RRLRTRSR-CONH₂ 323 ZElan058                           H₂N-K(dns)RRLRTR-CONH₂ 324 ZElan059                              H₂N-K(dns)rrLrTrSrPN-CONH₂ 325 ZElan073                          H₂N-K(dns)ASHNRRLRTR-CONH₂ 339 ZElan074                          H₂N-K(dns)SAHNRRLRTR-CONH₂ 340 ZElan075                          H₂N-K(dns)SSANRRLRTR-CONH₂ 341 ZElan076                          H₂N-K(dns)SSHARRLRTR-CONH₂ 342 ZElan077                          H₂N-K(dns)SSHNARLRTR-CONH₂ 343 ZElan078                          H₂N-K(dns)SSHNRALRTR-CONH₂ 344 ZElan079                          H₂N-K(dns)SSHNRRARTR-CONH₂ 345 ZElan080                          H₂N-K(dns)SSHNRRLATR-CONH₂ 346 ZElan081                          H₂N-K(dns)SSHNRRLRAR-CONH₂ 347 ZElan082                          H₂N-K(dns)SSHNRRLRTA-CONH₂ 348 SCRAMBLED PAX2PEPTIDES: ZElan083 H₂N-K(dns)GRNHDVVSSNTHKSYRSPRSASYPRLSNDRTDRTEPAPSS-CONH₂ 350 ZElan084 H₂N-K(dns)RNTRNKTSRLSANPHRSHR-CONH₂ 351

HAX42 Peptides—Relative Binding to P100 Fractions

ZElan021 (SEQ ID NO: 285), full length HAX42, was given the arbitraryvalue of 1.00 for binding to P100 at a given peptide concentrationdetermined from the signal-to-noise ratio data. HAX42 and itsderivatives are given as a ratio of HAX42 value to reflect their bindingabilities to P100 membrane fractions derived from a Caco-2 cell line asshown in Table 31. Table 32 provides a line-up of the HAX42 peptidesshowing the positive binding peptides in boldface. A core binding motifappears to lie in the amino acid sequence PGDYNCCGNCNSTG (ZElan091, SEQID NO:358).

TABLE 31 HAX42 Binding value Binding value Binding value Binding valueBinding value Binding value SEQ ID NO. peptide at 20 μg/ml at 50 μg/mlat 50 μg/ml at 25 μg/ml at 25 μg/ml at 25 μg/ml 285 ZElan021 1.00 1.001.00 1.00 1.00 1.00 326 ZElan060 0.44 0.56 0.43 327 ZElan061 0.20 0.600.38 328 ZElan062 0.11 0.42 0.34 331 ZElan065 0.00 0.54 0.30 333ZElan067 0.08 0.52 0.40 336 ZElan070 0.59 0.97 0.39 337 ZElan071 1.220.89 0.75 338 ZElan072 0.83 0.61 0.88 354 ZElan087 0.46 0.44 355ZElan088 2.21 1.41 1.63 356 ZElan089 0.55 0.44 0.49 357 ZElan090 2.061.54 2.16 358 ZElan091 2.02 1.37 1.20 359 ZElan092 1.41 1.90 0.91 360ZElan093 1.88 1.37 1.33

TABLE 32 HAX42 Peptide Amino acid sequence SEQ. ID. NO. ZElan021 H₂N-K(dns)SDHALGTNLRSDNAKEPGDYNCCGNGNSTGRKVFNRRRPSAIPT-CONH₂ 285 ZElan060H ₂N-K(dns)SDHALGTNLRSDNAKEPGDYNCCGNG-CONH₂ 326 ZElan061                      H₂N-K(dns)GNGNSTGRKVFNRRRPSAIPT-CONH₂ 327 ZElan062H₂N-K(dns)SDHALGTNLRSDNAKEPG-CONH₂ 328 ZElan065                              H₂N-K(dns)RKVFNRRRPS-CONH₂ 331 ZElan067                                  H₂N-K(dns)NRRRPS-CONH₂ 333 ZElan070 H₂N-K(dns)SDHALGTNLRSDNAKEPGDYNCCGNGNST-CONH₂ 336 ZElan071        H₂N-K(dns)NLRSDNAKEPGDYNCCGNGNSTGRKVFNR-CONH₂ 337 ZElan072                H ₂N-K(dns)PGDYNCCGNGNSTGRKVFNRRRPSAIPT-CONH₂ 338ZElan087 H₂N-K(dns)SDHALGTNLRSDNAKEPGDY-CONH₂ 354 ZElan088           H₂N-K(dns)SDNAKEPGDYNCCGNGNSTG-CONH₂ 355 ZElan089H₂N-K(dns)SDHALGTNLRSDNAK-CONH₂ -CONH₂ 356 ZElan090                H₂N-K(dns)EPGDYNCCGNGNSTG 357 ZElan091                 H₂N-K(dns)PGDYNCCGNGNSTG-CONH₂ 358 ZElan092                 H₂N-K(dns)PGDYNCCGNG-CONH₂ 359 ZElan093                     H₂N-K(dns)NCCGNGNSTG-CONH₂ 3609. Formulations

General Method for Preparation of Coacervated Particles.

Solid particles containing a Therapeutic as defined herein are preparedusing a coacervation method. The are particles are formed from a polymerand have a particle size of between about 10 nm and 500 μm, mostpreferably 50 to 800 nm. In addition the particles contain targetingligands which are incorporated into the particles using a number ofmethods.

The organic phase (B) polymer of the general method given above may besoluble, permeable, impermeable, biodegradable or gastroretentive. Thepolymer may consist of a mixture of polymer or copolymers and may be anatural or synthetic polymer. Representative biodegradable polymersinclude without limitation polyglycolides; polylactides;poly(lactide-co-glycolides), including DL, L and D forms;copolyoxalates; polycaprolactone; polyesteramides; polyorthoesters;polyanhydrides; polyalkylcyanoacrylates; polyhydroxybutyrates;polyurethanes; albumin; casein; citosan derivatives; gelatin; acacia;celluloses; polysaccharides; alginic acid; polypeptides; and the like,copolymers thereof, mixtures thereof and stereoisomers thereof.Representative synthetic polymers include alkyl celluloses; hydroxalkylcelluloses; cellulose ethers; cellulose esters; nitrocelluloses;polymers of acrylic and methacrylic acids and esters thereof; dextrans;polyamides; polycarbonates; polyalkylenes; polyalkylene glycols;polyalkylene oxides; polyalkylene terephthalates; polyvinyl alcohols;polyvinyl ethers; polyvinyl esters; polyvinyl halides;poyvinylpyrrolidone; polysiloxanes and polyurethanes and co-polymersthereof.

Typically, particles are formed using the following general method:

An aqueous solution (A) of a polymer, surface active agent, surfacestabilising or modifying agent or salt, or surfactant preferably apolyvinyl alcohol (PVA) or derivative with a % hydrolysis 50–100% and amolecular weight range 500–500,000, most preferably 80–100% hydrolysisand 10,000–150,000 molecular weight, is introduced into a vessel. Themixture (A) is stirred under low shear conditions at 10–2000 rpm,preferably 100–600 rpm. The pH and/or ionic strength of this solutionmay be modified using salts, buffers or other modifying agents. Theviscosity of this solution may be modified using polymers, salts, orother viscosity enhancing or modifying agents.

A polymer, preferably poly(lacide-co-glycolide), polylactide,polyglycolide or a combination thereof or in any enantiomeric form or acovalent conjugate of the these polymers with a targeting ligand isdissolved in water miscible organic solvents to form organic phase (B).Most preferably, a combination of acetone and ethanol is used in a rangeof ratios from 0:100 acetone: ethanol to 100:0 acetone: ethanoldepending upon the polymer used.

Additional polymer(s), peptide(s) sugars, salts, natural/biologicalpolymers or other agents may also be added to the organic phase (B) tomodify the physical and chemical properties of the resultant particleproduct.

A drug or bioactive substance may be introduced into either the aqueousphase (A) or the organic phase (B). A targeting ligand may also beintroduced into either the aqueous phase (A) or the organic phase (B) atthis point.

The organic phase (B) is added into the stirred aqueous phase (A) at acontinuous rate. The solvent is evaporated, preferably by a rise intemperature over ambient and/or the use of a vacuum pump. The particlesare now present as a suspension (C). A targeting ligand may beintroduced into the stirred suspension at this point.

A secondary layer of polymer(s), peptide(s) sugars, salts,natural/biological polymers or other agents may be deposited on to thepre-formed particulate core by any suitable method at this stage.

The particles (D) are then separated from the suspension (C) usingstandard colloidal separation techniques, preferably by centrifugationat high ‘g’ force, filtration, gel permeation chromatography, affinitychromatography or charge separation techniques. The supernatant isdiscarded and the particles (D) re-suspended in a washing solution (E)preferably water, salt solution, buffer or organic solvent(s). Theparticles (D) are separated from the washing liquid in a similar manneras previously described and re-washed, commonly twice. A targetingligand may be dissolved in washing solution (E) at the final washingstage and may be used to wash the particles (D).

The particles may then be dried. Particles may then be further processedfor example, tabletted, encapsulated or spray dried.

The release profile of the particles formed above may be varied fromimmediate to controlled or delayed release dependent upon theformulation used and/or desired.

Drug loading may be in the range 0–90% w/w. Targeting ligand loading maybe in the range 0–90% w/w.

Specific examples include the following examples:

EXAMPLE 1 Peptide Added at the Final Washing Stage

-   Product: Bovine Insulin loaded nanoparticles-   Aim: To prepare a 2 g batch of insulin loaded nanoparticles at a    theoretical loading of 50 mg/g and with the peptide ZElan018 (SEQ ID    NO: 281) added.

Formulation Details RG504H (Lot no. 250583) 2.0 g Acetone 45 ml Ethanol:5 ml PVA (aq. 5% w/v) 400 ml Bovine Insulin (Lot no. 86H0674) 100 mgPeptide: PAX2 (ZElan018, SEQ ID NO: 281) 10 mg/50 ml dH₂OExperimental Details:

The 5% w/v PVA solution was prepared by heating water until near boilingpoint, adding PVA and stirring until cool. The organic phase wasprepared by adding acetone, 45 ml, and ethanol, 5 ml, together. Thepolymer solution was prepared by adding RG504H, 2 g, to the organicphase and stirring until dissolved. The IKA™ reactor vessel was set up,all seals greased and the temperature was set at 25° C. The PVAsolution, 400 ml, was added into the reactor vessel and stirred at 400rpm.

Bovine insulin, 100 mg, was added into the stirring PVA solution. Usingclean tubing and a green needle, the polymer solution was slowly drippedin the stirring PVA solution with the peristaltic pump set at 40. Thesolvent was allowed to evaporate by opening the ports and allowing thedispersion to stir overnight at 400 rpm.

The suspension was centrifuged in a Beckman Ultracentrifuge™ withswing-out rotor at 12,500 rpm, 4° C. The supernatant was decanted anddiscarded. The “cake” of particles was broken up and dH₂O (200 mls) wasadded to wash the particles. The centrifugation and washing steps wererepeated twice.

The peptide solution, (ZElan018, SEQ ID NO: 281), 10 mg in 50 ml dH₂O)was prepared and added to the particles for a final washing stage. Thesuspended particles were centrifuged as before. The supernatant liquidwas decanted, the ‘cake’ broken up, and the particles were dried in thevacuum oven.

The particles were ground, placed in a securitainer and sent foranalysis. The weight of particles recovered was 1.45 g. A SEM showeddiscrete, reasonably spherical particles in the 300–500 nm size range.The potency was 49.2 mg/g (98.0% of label claim). Peptide loading was2.42 μg/mg (48.4% of label claim).

EXAMPLE 2 Peptide Added at the Beginning of Manufacture

-   Product: Bovine Insulin loaded nanoparticles-   Aim: To prepare a 2 g batch of insulin loaded nanoparticles at a    theoretical loading of 50 mg/g and with the peptide ZElan018 (SEQ ID    NO: 281) added at the beginning of manufacture.

Formulation Details RG504H (Lot no. 250583) 2.0 g Acetone 45 ml Ethanol:5 ml PVA (aq. 5% w/v) 400 ml Bovine Insulin (Lot no. 65H0640) 100 mgPeptide: PAX2 (ZElan018ii) 10 mgExperimental Details:

The 5% w/v PVA solution was prepared by heating water until near boilingpoint, adding PVA and stirring until cool. The organic phase wasprepared by adding acetone, 45 ml, and ethanol, 5 ml, together. Thepolymer solution was prepared by adding RG504H (polyactide-co-glycolide,Boehringer Ingelheim), 2 g, to the organic phase prepared in step aboveand stirring until dissolved. The IKA™ reactor vessel was set up, allseals greased and the temperature was set at 25° C. The PVA solution,400 ml, was added into the reactor vessel and stirred at 400 rpm.

Bovine insulin, 100 mg, was added into the stirring PVA solution. PAX2(ZElan018ii, 10 mg) was added to the stirring PVA solution. Using cleantubing and a green needle, the polymer solution was slowly dripped intothe stirring PVA solution with the peristaltic pump set at 40. Thesolvent was allowed to evaporate by opening the ports and allowing thedispersion to stir overnight at 400 rpm. The suspension was centrifugedin a Beckman Ultracentrifuge™ with swing-out rotor at 12,500 rpm, 4° C.The supernatant was decanted and discarded.

The “cake” of particles was broken up and dH₂O (200 ml) was added towash the particles. The centrifugation and washing steps were repeatedtwice. The ‘cake’ was broken up and the particles were dried in thevacuum oven.

The particles were ground, placed in a securitainer and sent foranalysis. The weight of the particles recovered was 1.6 g. The potencywas 47.3 mg/g (94.6% of label claim). Peptide loading was 1.689 μg/mg(33.8% of label claim).

EXAMPLE 3 Peptide Added 1 Hour Before Centrifugation

-   Product: Bovine Insulin loaded nanoparticles-   Aim: To prepare a 1 g batch of insulin loaded nanoparticles at a    theoretical loading of 50 mg/g and with the peptide ZElan018 (SEQ ID    NO: 281) added 1 hour before centrifugation.

Formulation Details RG504H (Lot no. 250583) 1.0 g Acetone 22.5 mlEthanol: 2.5 ml PVA (aq. 5% w/v) 200 ml Bovine Insulin (Lot no. 65H0640)50 mg Peptide: PAX2 (ZElan018, SEQ ID NO: 281) 5 mgExperimental Details:

The 5% w/v PVA solution was prepared by heating water until near boilingpoint, adding PVA and stirring until cool. The organic phase wasprepared by adding acetone, 22.5 ml, and ethanol, 2.5 ml, together. Thepolymer solution was prepared by adding RG504H, 1 g, to the organicphase prepared above and stirring until dissolved. The IKA™ reactorvessel was set up, all seals greased and the temperature was set at 25°C. The PVA solution, 200 ml, was added into the reactor vessel andstirred at 400 rpm.

Bovine insulin, 50 mg, was added into the stirring PVA solution. Usingclean tubing and a green needle, the polymer solution was slowly drippedin the stirring PVA solution with the peristaltic pump set at 40. Thesolvent was allowed to evaporate by opening the ports and allowing thedispersion to stir overnight at 400 rpm.

PAX2 ((ZElan018, SEQ ID NO: 281) 5 mg) was added to the stirringparticle suspension. After 1 hr, the suspension was centrifuged in aBeckman Ultracentrifuge™ with swing-out rotor at 12,500 rpm, 4° C. Thesupernatant was decanted and discarded. The “cake” of particles wasbroken up and dH₂O (200 ml) was added to wash the particles. Thecentrifugation and washing steps were repeated twice.

The ‘cake’ was broken up and the particles were dried in the vacuumoven. The particles were ground, placed in a securitainer and sent foranalysis. Potency was 20.75 mg/g (41.5% of label claim). Peptide loadingwas 1.256 μg/mg (25.12% of label claim).

EXAMPLE 4 Leuprolide Acetate Loaded Nanoparticles

-   Aim: To prepare a 3 g batch of leuprolide-acetate loaded    nanoparticles at a theoretical loading of 20 mg/g and with the    peptide ZElan024 (SEQ ID NO: 288) added.

Formulation Details RG504H (Lot no. 271077) 3.0 g Acetone 67.5 mlEthanol: 7.5 ml PVA (aq. 5% w/v) 600 ml Leuprolide acetate (Lot no.V14094) 60 mg Peptide: P31 (ZElan024, SEQ ID NO: 288) 15 mg/50 ml dH₂OExperimental Details:

The PVA solution was prepared and the organic phase was prepared byadding acetone, 67.5 ml, and ethanol, 7.5 ml, together. The polymersolution was prepared by adding RG504H, 3 g, to the organic phaseprepared above and stirring until dissolved. The IKA™ reactor vessel wasset up, all seals greased and the temperature was set at 25° C. The PVAsolution, 600 ml, was added into the reactor vessel and stirred at 400rpm.

Leuprolide acetate, 60 mg, was added into the stirring PVA solution.Using clean tubing and a green needle, the polymer solution, was slowlydripped in the stirring PVA solution with the peristaltic pump set at40. The solvent was allowed to evaporate by opening the ports andallowing the dispersion to stir overnight at 400 rpm. The suspension wascentrifuged in a Beckman Ultracentrifuge™ with swing-out rotor at 15,000rpm, 4° C. The supernatant was decanted and retained for analysis.

The “cake” of particles was broken up and dH₂O 200 ml) was added to washthe particles. The centrifugation and washing steps were repeated twice.

The peptide solution (P31 (SEQ ID NO:270), 15 mg in 50 ml dH₂O) wasprepared and added to the particles for a final washing stage. Thesuspended particles were centrifuged as before. The supernatant liquidwas decanted, and the particles were dried in the vacuum oven.

The particles were ground, placed in a securitainer and sent foranalysis. The weight of particles recovered was 1.87 g. SEM showeddiscrete, reasonably spherical particles in the 300–500 nm size range.The potency was 4.7 mg/g (23.4% of label claim). Peptide loading was1.76 μg/mg.

EXAMPLE 5 Peptide Added by ‘Spiking’ Polymer Phase with Polymer-PeptideConjugate

-   Product: Bovine Insulin loaded nanoparticles-   Aim: To prepare a 3 g batch of insulin loaded nanoparticles at a    theoretical loading of 50 mg/g and with the polymer-peptide    conjugate PLGA-ZElan019 added.

Formulation Details RG504H (Lot no. 271077) 2.85 g RG504H-ZElan019conjugate 0.15 g (5PAX5-conjugate) Acetone 67.5 ml Ethanol: 7.5 ml PVA(aq. 5% w/v) 600 ml Bovine Insulin (Lot no. 86H0674) 150 mgExperimental Details:

The 5% w/v PVA solution was prepared by heating water until near boilingpoint, adding PVA and stirring until cool. The organic phase wasprepared by adding acetone, 67.5 ml, and ethanol, 7.5 ml, together. Thepolymer solution was prepared by adding RG504H and the polymer-peptideconjugate to the organic phase and stirring until dissolved.

The IKA™ reactor vessel was set up, all seals greased and thetemperature was set at 25° C. The PVA solution, 400 ml, was added intothe reactor vessel and stirred at 400 rpm.

Bovine insulin, 100 mg, was added into the stirring PVA solution. Usingclean tubing and a green needle, the polymer solution, was slowlydripped in the stirring PVA solution with the peristaltic pump set at40. The solvent was allowed to evaporate by opening the ports andallowing the dispersion to stir overnight at 400 rpm.

The suspension was centrifuged in a Beckman Ultracentrifuge™ withswing-out rotor at 12,500 rpm, 4° C. The supernatant was decanted anddiscarded. The “cake” of particles was broken up and dH₂O (200 ml) wasadded to wash the particles. The centrifugation washing step wasrepeated twice.

The ‘cake’ was broken up and the particles were dried in the vacuumoven. The particles were ground, placed in a securitainer and sent foranalysis. The weight of particles recovered was 2.8 g. The potency was53.1 mg/g 106.2% of label claim). Peptide loading was 4.02 μg/mg (80.4%of label claim).

10. Animal Studies

Study 1

An open-loop study in which the test solution was injected directly intothe ileum was done. Wistar rats (300–350 g) were fasted for 4 hours andanaesthetized by intramuscular administration 15 to 20 minutes prior toadministration of the test solution with a solution of ketamine [0.525ml of ketamine (100 mg/ml) and 0.875 ml of acepromazine maleate-BP ACP(2 mg/ml)]. The rats were then injected with a test solution (injectionvolume: 1.5 ml PBS) intra-duodenally at 2–3 cm below the pyloris. Thetest solution contained either PLGA particles manufactured according tothe coacervation procedure given above with or without targetingpeptides or by the “spiked” method given above. Insulin (fast-actingbovine; 28.1 iu/mg) was incorporated in the particles at 5% drug loadingfor a total of 100 iu insulin (70 mg particles) or 300 iu insulin (210mg particles). Blood glucose values for the rats were measured using aGlucometer™ (Bayer; 0.1 to 33.3 m/mol/L); plasma insulin values weremeasured using a Phadeseph RIA Kit™ (Upjohn Pharmacia; 3 to 240μU/ml-assayed in duplicate). Systemic and portal blood was sampled.

Study groups included animals receiving test solutions containingparticles coated with the following peptides shown in Table 33.

TABLE 33 Study Group Receptor Peptide I hSI SNi10 SNi34 II hPEPT1 P315PAX5 III HPT1 PAX2 HAX42 IV D2H DCX8 DCX11 V (“spiked”) hPEPT1 P31-PLGAconjugate 5PAX5-PLGA conjugateControl groups included: 1) PBS control (1.5 ml) Open-Loop; 2) Insulinsolution (1 iu/0.2 ml) subcutaneous; 3) Insulin particles—no peptide (1iu/0.2 ml) subcutaneous; 4) Insulin particles/all 8 peptides mix (1iu/0.2 ml) subcutaneous; 5) Insulin loaded particles/peptide control(scrambled 5PAX5) (100 iu/1.5 ml) Open-Loop; 6) Insulin loadedparticles/peptide control (scrambled 5PAX5) (300 iu/1.5 ml) Open-Loop;7) Control particles (insulin-free)/all 8 peptide mix (equivalent 100iu/1.5 ml) Open-Loop; and 8) Control particles (insulin-free)/all 8peptide mix (equivalent 300 iu/1.5 ml) Open-Loop.

The following describes the pharmacokinetics for 300 iu-loading:

Target Receptor F %* Fold-increase** Stat. Sig.** HPT1 10.37 17.0 <0.001Spiked hPEPT1 4.94 7.5 0.005 PAX2 scrambled 3.50 3.6 NS Mix-8 2.00 2.0NS hPEPT1 1.60 1.5 NS D2H 1.57 1.4 NS hSI 0.54 0.9 NS *based on areaunder the curve (AUC) (1–4h), base-line adjusted, relative tosubcutaneous insulin solution liu **Fold increase in AUC compared toinsulin particles: 300iu

FIGS. 17A and 17B show the systemic blood glucose and insulin levelsfollowing intestinal administration of control (PBS); insulin solution;insulin particles; all 8 peptides mix particles and study grouppeptide-particles (100 iu). FIGS. 18A and 18B show the systemic bloodglucose and insulin levels following intestinal administration ofcontrol (PBS); insulin solution; insulin particles and study grouppeptide-particles (300 iu).

HPT1 targeted peptide coated particles provided the most potentenhancement of the delivery of insulin over subcutaneous injection ofinsulin followed by hPEPT1 spiked>PAX2scrambled>mix-8>hPEPT1>D2H>uncoated particles>hSI>solution. In a repeatstudy, the uncoated particles containing insulin gave similar profilesbut the HPT1-peptide targeted particles gave a reduced profile (3-fold).The insulin-free PLGA particles and the all-8 mix particles did not showan effect on the basal insulin or glucose levels. The HPT1 targetingparticles, the PEPT1 spiked, targeting particles, and the PEPT1targeting particles also reduced blood glucose levels indicative thatthe insulin delivered was bioactive. The other targeting particles werealso shown to reduce blood glucose levels although not to the sameextent as the HPT1 and PEPT1 spiked particles. No histologicaldifferences were observed in the small intestine for any of theformulations evaluated.

Study 2

A second open-loop study, similar to study 1 above, was undertaken withthe following treatment groups as shown in Table 34.

TABLE 34 Dose Group Insulin Number (iu) Description  1 PBS control  2a 1subcutaneous, bovine insulin  2b 2 subcutaneous, bovine insulin  2c 3subcutaneous, bovine insulin  2d 4 subcutaneous, bovine insulin  2e 10subcutaneous, bovine insulin  2f 20 subcutaneous, bovine insulin  2g 4subcutaneous, human insulin  3 300 uncoated insulin particles  4 100HAX42/PAX2 with 300 iu particle loading  5 300 HAX42/PAX2 (40mer)particles  6 300 HAX42 (40mer) particles  7 300 HAX42 particles +10-fold excess free HAX42 (40mer)  8 300 PAX2 (40mer) particles  9 300PAX2 freeze-dried (40mer) particles 10 300 PAX2 scrambled particles III(40mer) 11 300 PAX2 scrambled particles IV (19mer) 12 300 5PAX5/P31(40mer) particles 13 300 P31 (40mer) particles 14 300 5PAX5 (40mer)particles 15 300 HAX42 (27mer) particles 16 300 PAX2 (20mer) particles17 300 P31 (20mer) particles 18 300 PAX2 (15mer) particles 19 300 P31(15mer) particles 20 300 P31 D-form I(5 D-arginine)(16mer) particles 21300 P31 D-form II(2 D-arginine) (16mer) particles 22 300 HAX42 (10mer)

Availability of insulin following administration was assessed relativeto a 1 and 20 iu subcutaneous dose because the response to increasingsubcutaneous doses of bovine insulin does not increase linearly over therange of 1 to 20 iu. Data up to three hours post-dosing was availablefor most animals. Therefore, availability was first assessed usingindividual AUC(0–3h) data estimated from baseline-subtracted data forwhich data up to 3 hours was available. This approach may lead to anunderestimation of the availability as some animals that gave a highresponse often did not survive for 3 hours and, therefore, were excludedfrom the analyses. In an attempt to capture as much of these highresponses observed at the earlier timepoints as possible, the meanbaseline-subtracted plasma concentration data was used to estimate anAUC for each group. Table 35 shows the results based on this secondapproach (AUC(0–3h) calculated from the mean plasma concentration data).

TABLE 35 Group Dose iu Mean AUC_((0–3h)) F vs. 1 iu F vs. 20 iu  1 02.14  2a 1 875.27 100.00 28.86  2b 2 2439.36 139.35 40.22  2c 3 3671.44139.82 40.36  2d 4 6912.18 197.43 56.98  2e 10 27224.41 311.04 89.77  2f20 60651.28 346.47 100.00  2g 4 14255.49 407.17 117.52  3 300 10677.784.07 1.17  3 -Rat43 300 4645.06 1.77 0.51  4 100 3527.18 4.03 1.16  5300 27112.26 10.33 2.98  6 300 33091.68 12.60 3.64  7 300 9303.09 3.541.02  8 300 34241.83 13.04 3.76  9 300 10968.83 4.18 1.21 10 30027692.78 10.55 3.04 11 300 3004.29 1.14 0.33 12 300 18852.61 7.18 2.0713 300 20278.43 7.72 2.23 14 300 17400.38 6.63 1.91 15 300 16775.69 6.391.84 16 300 14217.47 5.41 1.56 17 300 8197.97 3.12 0.90 18 300 25050.599.54 2.75 19 300 7927.96 3.02 0.87 20 300 21519.57 8.20 2.37 21 3006322.41 2.41 0.69 22 300 12553.01 4.78 1.38The data for group 3 (uncoated insulin particles) are expressed with andwithout Rat 43. This animal had an atypically high response to theseuncoated particles and, therefore, may have biased the data for thisgroup.

This data shows that a combination of peptide-coated particles(HAX42/PAX2 or 5PAX5/P31) shows no greater availability than particlescoated with the individual peptides. Further, peptide-coated particleshave a greater availability than uncoated peptides. Scrambling the 40merPAX2 peptide did not result in a loss of bioavailability. Scrambling thePAX2 peptide and reducing the size to 19mer resulted in a loss ofbioavailability although this loss may be attributed in part to thereduction in peptide size. Reducing peptide size resulted in loss ofbioavailability. The D-form of P31 (ZElan053, SEQ ID NO: 319) hadincreased bioavailability possibly due to greater resistance to peptidebreakdown. A competitive excess of peptide resulted in a loss ofbioavailability, and freeze drying caused a loss in bioavailability. Byway of example, measurement of blood glucose levels showed that the HPT1and hPEPT1 targeting particles incorporating HAX42, PAX2, P31 (SEQ IDNO:270), and P31 D-form (ZElan053, SEQ ID NO: 319) reduced blood glucoselevels indicating that the insulin delivered was bioactive.

In further studies, insulin was recovered from the targeting particlesfollowing particle formation by dissolution and analyzed byelectrophoresis in non-denaturing sodum dodecyl sulfate (SDS)polyacrylamide gel electrophoresis (PAGE). The analysis of the insulinby non-denaturing SDS-PAGE and also by western blot transferred tomembranes and subsequent screening with an antibody to insulin,indicated that the insulin was intact, with no evidence of degradation,dimerization, or aggregation during the process of particle formation.

Study 3

An intraduodenal open loop model study was carried out on Wistar rats(300–350 g). Group 1 was administered leuprolide acetate (12.5 μg)subcutaneously. Group 2 was administered intraduodenally uncoatedleuprolide acetate particles (600 μg, 1.5 ml). Group 3 wasintraduodenally administered leuprolide acetate particles coated withPAX2 (600 μg; 1.5 ml). Group 4 was administered intraduodenallyleuprolide acetate particles coated with P31 (SEQ ID NO:270) (600 μg,1.5 ml). FIG. 19 shows the leuprolide plasma concentration followingadministration to these four groups. Both the P31 (SEQ ID NO:270) andthe PAX2 coated leuprolide particles administered intraduodenallyprovided enhanced plasma levels of leuprolide relative to subcutaneousinjection.

Homologies of GIT transport-binding peptides to known proteins are shownin FIGS. 20, 21A–F, and 22 A–D.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theappended claims.

Various publications are cited herein, the disclosures of which areincorporated by reference in their entireties.

1. A method of delivering a drug to a subject comprising administeringto the subject a pharmaceutical composition comprising a therapeuticallyeffective amount of a nucleic acid encoding a chimeric proteincomprising (i) a first protein comprising the amino acid sequence of SEQID NO: 51, said amino acid sequence being capable of specificallybinding to the gastro-intestinal receptor HPT1 (SEQ ID NO: 178), whichcomprises said first protein being fused via a covalent bond to a secondprotein, wherein the second protein acts as a drug; and (ii) apharmaceutically acceptable carrier.
 2. A method of delivering an activeagent in vivo comprising administering to a subject a compositioncomprising an isolated protein comprising the amino acid sequence of SEQID NO: 51, said amino acid sequence being capable of specificallybinding to the gastro-intestinal receptor HPT1 (SEQ ID NO: 178), whereinthe isolated protein is bound to a material comprising an active agentselected from the group consisting of an antigen, imaging agent anddrug.
 3. A method of delivering a drug to a subject comprisingadministering to the subject a composition comprising an isolatedprotein comprising the amino acid sequence of SEQ ID NO: 51, said aminoacid sequence being capable of specifically binding to thegastro-intestinal receptor HPT1 (SEQ ID NO: 178), wherein the isolatedprotein is covalently bound to a particle containing the drug.
 4. Amethod of delivering a drug to a subject comprising administering to thesubject a composition comprising an isolated protein comprising theamino acid sequence of SEQ ID NO: 51, said amino acid sequence beingcapable of specifically binding to the gastro-intestinal receptor HPT1(SEQ ID NO: 178), wherein the isolated protein is covalently bound tothe drug.
 5. The method of claim 2 wherein the active agent is a drug.6. The method of claim 2 wherein the material is a particle containingthe active agent.
 7. The method as in any one of claims 2 to 4 whereinthe isolated protein consists essentially of the amino acid sequence ofSEQ ID NO:
 51. 8. The method as in any one of claims 2 to 4 wherein theisolated protein facilitates the transport of the active agent throughhuman or animal gastro-intestinal tissue.
 9. The method as in any one ofclaims 1 to 4 wherein the administration is oral.
 10. The method as inany one of claims 1 to 4 wherein the subject is human.